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Fish Pathogen Monitoring Studies

Fish Pathogen Monitoring Studies

Ceratonova shasta in the Klamath River

Schematic showing the life cycle of Ceratonova shasta and influential factors

The life cycle of Ceratonova shasta and abiotic and biotic factors that influence the different stages. Published in: Bartholomew JL, Alexander JD, Hallett SL, Alama-Bermejo G, Atkinson SD (2022). Ceratonova shasta: a cnidarian parasite of annelids and salmonids. Parasitology 149, 1862–1875. https://doi.org/10.1017/S0031182022001275

Ceratonova shasta is a freshwater, myxozoan parasite that is native to the Pacific North West of North America. It causes enteronecrosis in juvenile salmonids and is associated with population-level impacts in the Klamath River. Transmission occurs through waterborne stages: actinospores released from annelids infect salmonid fishes and develop into myxospores which then infect annelids (see life cycle, above). The parasite proliferates in each host.

In response to the high prevalence and severity of C. shasta-infection in Klamath salmonids, we developed a parasite monitoring program to track the spatial and temporal abundance of C. shasta. The three main approaches are based on the parasite's life cycle and include sentinel fish exposures, annelid host sampling and molecular quantification of parasite DNA in water samples. These are described in more detail below. Monitoring occurs at established index sites which are shown on the following map.

Map of the lower Klamath River showing sampling sites

Klamath River Index Sites with site abbreviations and river kilometers (Rkm). Iron Gate Dam (Rkm 306) blocks anadromous salmonid migration.

Data shared here are preliminary and subject to modification.
Page photo credits: S Atkinson, S Hallett & J Alexander
Monitoring Studies are Primarily Supported by the Bureau of Reclamation.

Sentinel fish exposures

In sentinel fish exposures, fish highly susceptible to the parasite (out-of-basin rainbow trout) are placed in cages alongside fish of interest such as in-basin Chinook and coho salmon at index sites along the river for a three day exposure. All fishes are transported to OSU's John L Fryer Aquatic Animal Health Lab and monitored for infection (~ 60 days). Severity of infection (percent morbidity and mean days to morbidity) are recorded through visual observations and molecular assay (PCR).

Photo of juvenile fish being added to a cage in the river (left side) and the cage submerged in the river (right side)

Juvenile fish being placed in a sentinel cage which is then submerged in the Klamath River

2024 data updates

An April sentinel fish exposure occurred 04/18 - 04/21 at two mainstem sites, KBC and KSV, with Fall Creek Hatchery Fall Chinook and out-of-basin rainbow trout.

A May sentinel fish exposure will occur 05/20 - 05/23 at WMR and KED in the upper basin and at KI5, KBC, KSV and KOR in the lower basin.

A third sentinel fish exposure is scheduled to occur at all index sites in June, 2024.

An exposure is scheduled to occur in September at two lower basin sites.

Water samples

To detect and quantify waterborne stages of C. shasta, river water samples are collected at five mainstem index sites; once a week all year round at two sites (KBC and KSV) and once per week from April through October at three other sites. Solar-powered automatic samplers (ISCOs) collect 1L water every 2 hours for 24 hours, from which 4 1L samples are manually taken (see photos below). Each 1L sample is filtered through a nitrocellulose membrane using a vacuum pump, and any captured DNA in 3 of the replicate samples is extracted using a kit (see images on right). A quantitative PCR (qPCR) specific for C. shasta is used to detect and quantify any parasite DNA present. Cq values generated by the qPCR are converted to numbers of parasite spores per liter of water using reference samples with known quantities of spores. The Karuk and Yurok tribal biologists are integral to the collection and filtration of the water samples. Water samples are also taken in conjunction with the sentinel fish exposures; manual 'grab' samples are collected on the first and last day of the exposure. Data are presented as the average spores per liter of three replicate 1 liter water samples collected at each site and time.

Genotyping

There are multiple genetic types or genotypes of C. shasta simultaneously present in the Klamath River. These differentially disease the various salmonid species. For example, type I causes mortality in Chinook salmon whereas type II can be fatal for coho salmon. Type 0 is found in sympatric Oncorhynchus mykiss (steelhead and redband rainbow trout).

Therefore, we also genotype each water sample. This is done in two ways. For genotyping type II only, we use a qPCR that amplifies the variable ITS1 region specific to that genotype. Whereas, to determine the overall genotype composition of a sample, we amplify the variable ITS1 region common to all genotypes in a PCR and then sequence that amplicon. From the sequencing chromatogram, we can determine the proportion of each genotype present in a sample. We use the total spore density to then determine the number of spores of each genotype in a sample.

Genotyping commences once we detect greater than 1 spore per liter.

Photo of person filtering a water sample using a vacuum pump (left side) which is then folded (right side)

Filtering a water sample using a vacuum pump (left). Folding the filter paper with captured material (right).

2024 data updates

We are still in the Winter collection phase, with only two Klamath River sites sampled each week: KSV & KBC. OSU has received and analyzed water samples up to and including 2/26/2024.

Samples were taken either by ISCO automatic sampler "i" and are given as the average density of C. shasta spores per liter of river water (from 3x 1-L subsamples of a 24-hour composite sample, Sunday-Monday), or taken manually "g" (3 x 1-L samples taken Monday).

No C. shasta DNA has been detected in water, which is not unusual during winter.

Specific results for 2 sites, '-' = no C. shasta DNA detected. If inhibition was detected this is specified. At the end of January and early February the high suspended sediment load caused inhibition of the molecular assay.

01/02/2023
KBC -
KSV -

01/08/2023
KBC - (slight inhibition)
KSV no sample

01/15/2023
KBC - (slight inhibition)
KSV - (totally inhibited)

01/22/2023
KBC - (no inhibition)
KSV - (no inhibition)

01/29/2023
No samples - sediment load too high

02/05/2023
No samples - sediment load too high

02/12/2023
No samples - sediment load too high

02/19/2023
KBC -
KSV -

02/26/2023
KBC -
KSV -

C. shasta DNA was detected in only one subsample collected at KBC and KSV during these three weeks.

Specific results for 2 sites, '-' = no C. shasta DNA detected. If inhibition was detected this is specified.

Sampling at all index sites will commence late March.

03/04/2024

KBC trace (in 1/3 samples)

KSV -


03/12/2024

KBC -

KSV -


03/18/2024

KBC -

KSV -

This was the first week of sampling at all 6 index sites. Multiple sites required double the regular number of filters due to high levels of suspended material.

No C. shasta DNA was detected at any site this week.

03/25/2024

KSH - (substitute site for KI5)

KBC -

KMN -

KSV -

KOR -

KTC -

Due to the higher than usual amount of suspended material in the water column, which renders filtering challenging, less water was filtered at most sites this week (volume indicated). ISCO = automatic water sampler; grab = manual sample. '-' = no C. shasta DNA detected

No C. shasta DNA detected was detected at any site this week, however, the sensitivity of the approach is affected by the high river turbidity.

04/01/2024

KSH - (100ml; grab)

KBC - (100ml; isco)

KMN - (100ml; grab)

KSV - (100ml; isco)

KOR - (250ml; isco)

KTC - (1L; grab)

This was the first week of sampling at all index sites, with expedited sample processing. Multiple sites required a reduction in water volume filtered due to high levels of suspended material. Two sites were sampled with an automatic sampler ("i"), four sites were a manual grab ("grab")(water levels were too high at KTC to deploy an ISCO).

Only trace amounts of Cs-DNA was detected at any of the 6 sites analyzed (at KTC):

04/08/2024

KSH - (100ml; isco)

KBC - (100ml; grab)

KMN trace (1/3 samples positive; 100ml; isco)

KSV - (100ml; grab)

KOR - (250ml; grab)

KTC - (1L; grab)

​This was the 2nd week of sampling at all index sites, with expedited sample processing. C. shasta DNA was detected at 5 of the 6 index sites (in at least one of three replicate samples). Because of the higher than usual levels of suspended substrate, subsamples of less than 1L (100 - 350mL) were filtered for each site except the lowermost, KTC.

Estimated spores/L based on low-volume subsamples (reduced owing to ongoing high sediment loads):

04/15/2024

KSH 2/3 samples positive; 100mL; isco; estimated 3 sp/L

KBC 2/3 samples positive; 100mL; isco; estimated 6 sp/L

KMN 3/3 samples positive; 100mL; isco; estimated 5 sp/L

KSV 1/3 samples positive; 100mL; grab; estimated <1 sp/L

KOR 3/3 samples positive; 350mL; isco; estimated 4 sp/L

KTC 0/3 samples positive; 1L; grab; negative

We will test 1 replicate sample with > 5 spores/L from each site for genotype II (coho-relevant). Results: KOR trace detection, KSH, KBC, KMN no detection.

​This was the 3rd week of sampling at all index sites, with expedited sample processing. We received samples from 5 of the 6 sites (KTC shipping was delayed). Because of the higher than usual levels of suspended substrate this season, subsamples of less than 1L (150 - 300mL) were filtered for each site.

C. shasta DNA was detected at all 5 sites assessed (in all three of the three replicate samples per site), at higher density than the previous week. Average spore density ranged from 7 spores per liter (at KSV and KOR) up to 57 spores per liter (at KBC). Spore density exceeded 10 spores per liter (Chinook 40% mortality threshold) at 3 sites: the 3 most upstream, KSH, KBC and KMN.

All samples were also assessed for coho type II this week. Trace amounts were detected at 3 of the 5 index sites.

SITE, number of replicates positive, sample volume, method of collection, average total spore density (followed by coho type II results):

KSH 3/3 samples positive; 150ml; isco; estimated 13 sp/L

Type II "coho": 1/3 samples positive, trace sp/L

KBC 3/3 samples positive; 150mL; isco; estimated 57 sp/L

Type II "coho": 1/3 samples positive, trace sp/L

KMN 3/3 samples positive; 150ml; isco; estimated 22 sp/L

Type II "coho": 0/3 samples positive

KSV 3/3 samples positive; 200ml; grab; estimated 7 sp/L

Type II "coho": 1/3 samples positive, trace sp/L

KOR 3/3 samples positive; 300ml; isco; estimated 7 sp/L

Type II "coho": 0/3 samples positive

KTC 3/3 samples positive; 1000ml; isco; estimated 3 sp/L

Type II "coho": 0/3 samples positive

​This was the 4th week of sampling at all index sites, with expedited sample processing. Because of the higher than usual levels of suspended substrate this season, subsamples of less than 1L (150 - 400mL) were filtered for each site except KTC (1L).

All samples tested positive for C. shasta this week: C. shasta DNA was detected at all 6 sites assessed, in all three of the three replicate samples per site. Density was higher than the previous week at 4 sites, lower at 2. Average spore density ranged from 2 spores per liter (at KTC) up to 46 spores per liter (at KBC). Spore density exceeded 10 spores per liter (Chinook 40% mortality threshold) at 4 sites (one more than the previous week): the 4 most upstream, KSH, KBC, KMN and KSV.

All samples were also assessed for coho type II. The density of type II was higher throughout the lower basin this week. Type II was detected at 5 of the 6 index sites (all except KTC), from < 1 spore to 3 spores per liter (at KBC).

SITE, number of replicates positive, sample volume, method of collection, average total spore density (followed by coho type II results):

KSH 3/3 samples positive; 200ml; isco; estimated 29 sp/L

2 sp/L type II "coho"

KBC 3/3 samples positive; 150mL isco; estimated 46 sp/L

3 sp/L type II "coho"

KMN 3/3 samples positive; 150ml; isco; estimated 36 sp/L

1 sp/L type II "coho"

KSV 3/3 samples positive; 200ml; grab; estimated 42 sp/L

2 sp/L type II "coho"

KOR 3/3 samples positive; 400ml; isco; estimated 9 sp/L

<1 sp/L type II "coho"

KTC 3/3 samples positive; 1000ml; isco; estimated 2 sp/L

no type II "coho"

​This was the 5th week of sampling at all index sites, with expedited sample processing. Because of the higher than usual levels of suspended substrate this season, subsamples of less than 1L (200 - 650mL) were filtered for each site except KTC (standard 1L); greater volumes were able to be filtered this week than last week though.

All samples tested positive for C. shasta this week, but at much reduced spore densities than last week. No inhibition was detected and results have been corrected for volume. The highest density of 4 spores per liter was measured at three sites (KSH, KBC and KSV). Spore density did not exceed the Chinook 40% mortality threshold (of 10 spores per liter) at any site.

All samples were also assessed for coho type II. The density of type II was similar throughout the lower basin this week as last week. Type II was detected at 4 of the 6 index sites (not KTC or KMN), from < 1 spore to 2 spores per liter (at KBC).

05/06/2024

KSH 3/3 samples positive; 200ml; isco; estimated 4 sp/L

1 sp/L type II "coho"

KBC 3/3 samples positive; 200mL isco; estimated 4 sp/L

2 sp/L type II "coho"

KMN 3/3 samples positive; 200ml; isco; estimated 2 sp/L

0 sp/L type II "coho"

KSV 3/3 samples positive; 300ml; grab; estimated 4 sp/L

1 sp/L type II "coho"

KOR 3/3 samples positive; 650ml; isco; estimated 3 sp/L

<1 sp/L type II "coho"

KTC 3/3 samples positive; 1000ml; grab; estimated <1 sp/L

no type II "coho"

​This was the 6th week of sampling at all index sites, with expedited sample processing. Because of the higher than usual levels of suspended substrate this season, subsamples of less than 1L (200 - 450mL) were filtered for each site except KTC (standard 1L).

All samples tested positive for C. shasta this week. The highest density of 25 spores per liter was measured at KBC. Spore density exceeded the Chinook 40% mortality threshold (of 10 spores per liter) at 5 of 6 sites (all except lowermost site, KTC).

All samples were also assessed for coho type II. The density of type II in the lower basin was higher than last week. Type II was detected in all samples for the first time this week, from < 1 spore to 3 spores per liter (at KBC & KSH); .

SITE, number of replicates positive, sample volume, method of collection, average total spore density (followed by coho type II results):

KSH 3/3 samples positive; 200ml; isco; estimated 15 sp/L

3 sp/L type II "coho"

KBC 3/3 samples positive; 200mL isco; estimated 25 sp/L

3 sp/L type II "coho"

KMN 3/3 samples positive; 250ml; isco; estimated 14 sp/L

2 sp/L type II "coho"

KSV 3/3 samples positive; 350ml; grab; estimated 16 sp/L

2 sp/L type II "coho"

KOR 3/3 samples positive; 450ml; isco; estimated 11 sp/L

2 sp/L type II "coho"

KTC 3/3 samples positive; 1000ml; isco; estimated 3 sp/L

<1 sp/L type II "coho"

Graph showing the density of waterborne Ceratonova shasta at the Beaver Creek index site in 2024.

Density (average spores per liter) of Ceratonova shasta in 24-hour composite water samples collected at the mainstem long term index site, near Beaver Creek (KBC) in 2024 (blue data points) compared to the previous years (2020-2023).

Graph showing the density of waterborne Ceratonova shasta at the index sites in the lower Klamath basin in 2024.

Density (average spores per liter) of Ceratonova shasta in 24-hour composite water samples collected at the mainstem index sites in 2024. Note that KMN is sampled only during salmonid outmigration, KBC and KSV year round and remaining sites April through October. KBC = near Beaver Creek, KSV = Seiad Valley, KSH = near Shasts River, KTC = Tully Creek, KMN = Kinsman Fish Trap, KOR = Orleans. The line denotes 10 spores per liter which corresponds with 40% mortality threshold in Chinook salmon.

Graph showing density of waterborne stages of the myxozoan parasite Ceratonova shasta in water samples collected at six index sites in the Klamath River.

Density (average spores per liter) of Ceratonova shasta in 24-hour composite water samples collected at the mainstem index sites in 2023. Note that KMN is sampled only during salmonid outmigration, KBC and KSV year round and remaining sites April through October. KBC = near Beaver Creek, KSV = Seiad Valley, KI5 = near I5 bridge, KTC = Tully Creek, KMN = Kinsman Fish Trap, KOR = Orleans. The line denotes 10 spores per liter which corresponds with 40% mortality threshold in Chinook salmon.

Scientist performing water sampling

Manual water sampling by Karuk Tribal Biologist Larry Alameda at index site K-I5

Scientist with solar powered automatic sampler

Solar powered automatic sampler at the Kinsman index site. An ISCO is used at all index sites but manual sampling provides a backup.

Scientist with water samples on truck bed

Collection of 4 1L technical replicate water samples from the larger 12L composite collected over 24 hours at KSV

Scientist sampling water from river

Manual water sampling at Orleans index site.

Photos courtesy of Karuk Tribal Biologist Larry Alameda

Annelid sampling

To monitor abundance and prevalence of C. shasta infection in the invertebrate annelid host, Manayunkia occidentalis, benthic samples are collected annually at seven sites in the Klamath River. Sites span a discharge gradient; 2 are located in the upper basin downstream from Keno Dam, 3 are located in the middle basin downstream from Iron Gate Dam, and 2 are located in the lower basin downstream from the Scott River. Samples are routinely collected in fall, winter, spring, and summer, and are scheduled to occur more frequently if flooding or pulse flow events are planned. Samples are processed to determine density, simple demographics, and prevalence of C. shasta infection.

A moss sample on a ruler in a hand.

Manayunkia occidentalis tubes from the image on right

A ruler sticking into a large sample of moss.

A high density assemblage sample at TOH

Diver collecting underwater sample

Diver collecting a benthic sample from boulder substrate at the TOH reach. We use a modified Hess sampler fitted with 80um Nytex mesh netting and a 500mL Nalgene collection bottle. Samples are preserved in ethanol until processing.

Benthic samples

Benthic samples are fractioned prior to sorting.

Diver photographing annelids

Dr. Alexander photographing annelids on riprap substrate at the KBC monitoring site.

Divers in the river

Annelid sampling at the KI5 reach. Foreground diver measures depth and velocity, while background diver measures substrate grain sizes.

Annual reports

Annual reports for Bureau of Reclamation funded studies for 2015 onwards are available. Please contact Sascha Hallett (halletts@oregonstate.edu). Annual reports are submitted June 1 the year after the research year.

Annual Klamath River Fish Health Workshop

We are pleased to announce this year's Annual Klamath River Fish Health Workshop to be held in Yreka, on Tuesday May 21st. We gratefully acknowledge our Karuk collaborators for hosting us at their Karuk Tribe Housing Authority, Yreka Apsuun Community Center.

This year, the KRFHW will precede the Spring KBMP meeting to be held Wednesday May 22nd at the same venue (please see emails from Randy Turner for KBMP specifics).

The general KRFHW schedule will be as follows:

Workshop: Tuesday May 21st (9am-5pm). Full day, public forum during which Klamath River fish health research updates, overviews, and presentations will be shared with an open forum Q & A session. Presentations will be basin-wide. Plans for 2024 and beyond will also be shared. The May 21 workshop will be open to the public to promote an open exchange of information, and attendance for all is free, however, we do request that you register your intention to attend on this spreadsheet.

The meeting format will be in-person. The agenda can be found here.

Project Planning Session: Wednesday May 22nd (8:30am-11:30am). Half day, smaller meeting to coordinate research efforts and management needs. The Project planning session is reserved for fish health researchers to discuss 2024 and beyond study plans and to promote project coordination and collaboration. The planning session will include an opportunity for Q & A between fish health researchers and agency/tribal co-managers. The meeting format will be in-person.

We look forward to seeing many familiar faces at this annual workshop on fish health related research in the Klamath River Basin!