Fish Pathogen Monitoring Studies
Ceratonova shasta in the Klamath River
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.
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).
2022 data updates
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.
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).
| KBC | KSV |
| 02/13/2023 | i 0 | i 0 |
| 02/06/2023 | g 0 | g 0 |
| 01/30/2023 | g 0 | i 0 |
| 01/23/2023 | g 0 | g 0 |
| 01/17/2023 | i 0 | g 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.
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.
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.
