Skip to main content

Fish Pathogen Monitoring Studies

Ceratonova shasta in the Klamath River

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

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

2022 data updates

An April sentinel fish exposure occurred 04/18 - 04/21 at KBC and KSV with IGH Fall Chinook and out of basin rainbow trout.

A May sentinel fish exposure occurred 05/19 - 05/22 at WMR and KED in the upper basin and at KI5, KBC, KSV and KOR in teh lower basin.

A third sentinel fish exposure occurred June 13–16.

A September exposure occurred 09/26 - 09/29.

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).

2023 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 2/13/2023.

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).

Samples from late January and into February have had fewer contaminants (sediment, organics) meaning less assay inhibition, although a low level of inhibition persists in most samples - likely due to continued runoff from fire damaged areas.

No C. shasta DNA has been detected in water since trace detections over the New Year. These very low to no detections of C. shasta DNA are typical of early-mid winter samples every year.

Specific results for 2 sites, spores/L, and indicating whether the sample was taken by automatic ISCO sampler (i) or manual grab (g).

KBCKSV
02/13/2023i 0i 0
02/06/2023g 0g 0
01/30/2023g 0i 0
01/23/2023g 0g 0
01/17/2023i 0g 0

No C. shasta DNA was detected in samples collected at KBC and KSV.

Sampling at all index sites will commence late March/early April.

Graph showing density of waterborne stages of teh myxozoan parasite Ceratonova shasta in water samples collected near Beaver Creek in the Klamath River.
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 2023 (blue data points) compared to 2022 (gray line).
An image of a scatterplot chart comparing Ceratonova shasta average spore densities at all index sites in 2022 with Ceratonova shasta spores per liter.
Density (average spores per liter) of Ceratonova shasta in 24-hour composite water samples collected at the mainstem index sites in 2022. 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.
Graph showing Ceratonova shasta average spore densities at all index sites in 2021
Density (average spores per liter) of Ceratonova shasta in 24-hour composite water samples collected at the mainstem index sites in 2021. 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 threhold in Chinook salmon.
Chart of Ceratonova shasta average spore densities in 2021
Density (average spores per liter) of Ceratonova shastagenotype II in 24-hour composite water samples collected at the mainstem index sites in 2021. 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 5 spores per liter which corresponds with a 40% mortality threhold in coho 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

The next workshop is TBD.