LONG-TERM FISH DISEASE MONITORING PROGRAM IN THE LOWER KLAMATH RIVER
Annual Report for 2010
Investigator: Jerri Bartholomew, Department of Microbiology, Oregon State University
Funding: Bureau of Reclamation
Objective 1: DEVELOPMENT OF A MULTIYEAR DATASET ON CERATOMYXA SHASTA INFECTION PREVALENCE
IN BOTH HOST POPULATIONS (FISH AND POLYCHAETE) AT SELECTED LOCATIONS TO MONITOR HOW
CHANGES IN FLOW, TEMPERATURE AND OTHER VARIABLES ALTER PARASITE INFECTION RATE.
Task 1.1 SELECTION OF INDEX SITES
Fish were not held at Tully Creek in 2010 but access was possible for water sampling. The remaining
index sites were consistent with 2009. The Williamson River site is located at the Nature Conservancy
and the Klamathon site is at the Klamath River Country Estates near Klamathon Bridge (Figure 1.1.1).
FIGURE 1.1.1. Map of Klamath River index sites for exposures
of sentinel fish (except Tully Creek) and collection of water
samples in 2010 with site abbreviations and river kilometers
(Rkm).
Seiad Valley
(KSV)
Rkm 207
Iron Gate Dam
Rkm 306
Above Beaver
Creek (KBC)
Rkm 258
Tully Creek
(KTC)
Rkm 62
Williamson
River (WMR)
Rkm 441
Keno Eddy
(KED)
Rkm 369
Klamathon
(KKB)
Rkm 302
Orleans
(KOR)
Rkm 90
Task 1.2 SENTINEL FISH EXPOSURES
Sentinel exposures in 2010 were conducted following the same protocol used in previous years to
determine:
1. How infection levels this year compare with levels in previous years.
2. If the distribution of the parasite has changed.
3. The relative susceptibility of Klamath River Chinook and coho salmon.
4. The effects of post-exposure water temperature on disease progress in Chinook and coho
salmon.
5. The relationship between parasite numbers measured in water samples and biological
effects in the different fish species.
1
Methods
In 2010, 72 hr river exposures were conducted during the months of April, May, June and September.
The April 23-26 exposure occurred in the Klamath River near Beaver Creek only. Exposures during
May 18-21 and June 15-18 occurred at 6 locations in the upper and lower river (Figure 1.1.1). The
September 20-23 exposure occurred in the Klamath River near Beaver Creek and at Seiad Valley.
Additionally, during May, the lower Shasta River was tested for the presence of C. shasta.
A known C. shasta-susceptible rainbow trout stock from Roaring River Hatchery (Oregon Department
of Fish and Wildlife) and Klamath River fall Chinook and coho salmon from Iron Gate Hatchery
(California Department of Fish and Game) were held at all sites with the exception that coho salmon
were not exposed at Keno Eddy in the upper Klamath River and only rainbow trout were exposed in
the Shasta River. Generally, 40 fish of each stock were held in live cages at each site. After exposure,
each group of fish was brought to the Salmon Disease Laboratory (SDL), Corvallis, Oregon, held in well
water at water temperatures similar to river temperatures during the 72 hr exposure and observed
for disease signs for at least 60 days. The fish were given preventative treatments for external
parasites and columnaris disease. In April, the sentinel fish were approximately 0.5-1.5 g size, May
1.5-2.5 g, June 2-9 g, and September 8-35 g. Fish were reared at either 13°C or 18°C, depending on
the site-specific river temperature during exposure. In 2010, in April and May the river exposure
water temperature was about 13°C and for June and September it was 18°C. All moribund fish were
evaluated for C. shasta infection by microscopic examination of a sample from their lower intestine
for the presence of myxospores. A subsample of moribund fish from each group was necropsied for
other parasite and bacterial infections. PCR testing for C. shasta occurred for fish that were negative
by microscopic examination.
The effect of water temperature on C. shasta infections in juvenile Chinook and coho salmon was also
studied. During the April (Chinook only), May, June, and September exposures in the Klamath River
near Beaver Creek, 80 fish of each stock (60 coho in September) were exposed and then each divided
into 2 tanks upon return to the SDL, one receiving water at 13°C and the other at 18°C. These fish
were reared for 60 days and clinical disease monitored as described above.
Results & Discussion
Average water temperatures during the 72 hr exposures are shown in Table 1.2.1. Water
temperatures in May 2010 were 1-2°C cooler at most sentinel sites than in May 2009.
TABLE 1.2.1. Average Klamath River water temperatures (°C) at sentinel sites during the 72-hour fish exposures
in 2010.
Site
Lower Williamson Rv
Keno Eddy
Klamathon
Near Beaver Creek
Seiad Valley
Orleans
April 23-26
12
May 18-21
13
13
13
14
11
10
June 15-18
16
17
18
18
15
14
September 20-23
17
17
Results of the sentinel exposures in April, May, June, and September are shown in Figures 1.2.2-1.2.8.
The values of percent loss represent fish that were moribund and in which myxospores of C. shasta
were observed, or, which were PCR positive for this parasite.
2
April 2010 sentinel exposure - The April 23-26 exposure of rainbow trout, IGH fall Chinook and coho
salmon juveniles near Beaver Creek followed by post-exposure holding in well water at 13°C resulted
in no loss of Chinook or coho, but 77% of the rainbow trout died and were infected with C. shasta.
However, Chinook held at 18°C post-exposure suffered 17% loss (Figure 1.2.5). Thus, if river water
temperatures had increased rapidly in May, Chinook that had received a lethal dose of C. shasta in
late April would have died. In 2009, IGH Chinook exposed in April then reared at 13°C incurred a 17%
loss associated with C. shasta, indicating that infection was more severe that year than in April of
2010. Rainbow trout held at 18°C post-exposure incurred 90% loss.
May 2010 sentinel exposures - The May 18-21 sentinel exposures of the susceptible rainbow trout,
IGH Chinook and coho salmon were conducted at the same 6 sentinel sites used in 2009. During this
exposure, water temperatures were cooler than those encountered in previous years in May, so postexposure rearing was at 13°C. The occurrence (Figure 1.2.2) of C. shasta in rainbow trout
demonstrated the wide distribution of infectious parasite in the Klamath River. Similar results were
observed in previous years, with high loss of the rainbow trout exposed in the lower Williamson River
and in the Klamath River from near Beaver Creek and downriver to Seiad Valley. At Keno Eddy and
near Klamathon Bridge, only 3% and 2% of the rainbow trout died respectively with C. shasta. At
Orleans, only 33% of the rainbow trout died with C. shasta in 2010 compared to 100% in May 2009. It
appears the cooler water temperatures in 2010 resulted in lower losses of the susceptible rainbow
trout at Keno Eddy, Klamathon and Orleans in May.
Sentinel rainbow trout held in the lower Shasta River then held post-exposure in the lab for 60 days
did not suffer any loss from C. shasta and therefore the infective stage of the parasite was not
detected in this tributary of the Klamath River.
For the IGH fall Chinook and coho salmon exposed in May, no C. shasta infections were observed at
any site except Seiad Valley and Keno Eddy where 2.6% of the IGH Chinook were infected with C.
shasta. This level of mortality was less at most sites in 2010 than in previous years. The finding of
one infected Chinook at Keno Eddy is the first time any Chinook has been found positive for C. shasta
at this site; this fish was visually negative but PCR positive.
FIGURE 1.2.2. Percent loss with C.
shasta infection of rainbow trout
(Rbt), IGH fall Chinook and coho
salmon exposed May 18-21, 2010 at
six sentinel sites in the Klamath River
basin and held for 60 days postexposure. See Figure 1.1.1 for site
abbreviations. (only Chinook and
rainbow trout were held at Keno
Eddy).
3
June 2010 sentinel exposures - C. shasta-associated mortality following the June 15-18 sentinel
exposure was higher at most sites than in May (Figure 1.2.3). Post-exposure rearing water
temperature was 18°C. The rainbow trout suffered high losses (>90%) at all sites except Keno Eddy
(45%) and near Klamathon Bridge (2.4%). In the upper river, no IGH Chinook or coho died after
exposure in the lower Williamson River or Keno Eddy (coho were not tested). Above Klamathon
Bridge at the Klamath River Country Estates site no Chinook or coho salmon died. Loss of IGH
Chinook occurred near Beaver Creek (20%), Seiad Valley (12.5%) and at Orleans (2.4%). Loss of coho
at Beaver Creek was 10%, Seiad Valley 5% and at Orleans 0%. The sentinel exposure results for June
2010 were similar to previous years in that most of the losses from C. shasta that occurred were in
the “infectious zone” i.e., near Beaver Creek and Seiad Valley but the total percent losses were much
lower.
FIGURE 1.2.3. Percent loss
with C. shasta infection of
rainbow trout, IGH fall
Chinook and coho salmon
exposed June 15-18, 2010 at
six sentinel sites in the
Klamath River basin and held
for 90+ days post-exposure.
See Figure 1.1.1 for site
abbreviations. Sites are listed
upstream (right) to
downstream (left).
September 2010 sentinel exposures - The susceptible rainbow trout, IGH fall Chinook and coho were
exposed September 20-23 at two sites: near Beaver Creek and Seiad Valley. Post-exposure rearing
temperature was 18°C. High losses from C. shasta occurred in the rainbow trout at both sites (Figure
1.2.4). No mortality was observed in IGH Chinook at either site. Coho salmon mortality with C. shasta
infection occurred only at Seiad Valley (6.7% loss). In September, the IGH fall Chinook were less
affected by C. shasta compared to June and the coho similarly at Seiad Valley.
FIGURE 1.2.4. Percent loss with C. shasta
infection of rainbow trout, IGH fall Chinook
and coho salmon exposed September 20-23,
2010 at six sentinel sites in the Klamath River
basin and held for 60+ days post-exposure.
See Figure 1.1.1 for site abbreviations.
4
Effect of post-exposure rearing water temperature on infections of C. shasta in Klamath River fish
stocks - Results of the comparison of percent loss of the susceptible rainbow trout, IGH Chinook and
coho with C. shasta infection when exposed near Beaver Creek in April, May, June, and September
and held at the laboratory at 2 different water temperatures are shown in Figure 1.2.5. The known
susceptible rainbow trout stock consistently suffered high loss even at the lower temperature of 13°C
for all months. For the IGH Chinook, post-exposure high water temperature of 18°C resulted in loss
with C. shasta infection in April (17%), May (16%) and June (20%) while at 13°C no loss was observed
during any month. For the coho, losses with C. shasta infection were only observed at the higher
temperature of 18°C for May (16%) and June (10%). The higher post-exposure rearing temperature of
18°C resulted in some increased loss from C. shasta, but even at this high temperature loss in 2010
was much lower than in previous years. Previous years’ studies indicated that coho loss from C.
shasta was affected more by post-exposure higher rearing temperature than the IGH Chinook.
FIGURE 1.2.5. Effect of post-exposure water temperature on percent C. shasta-associated mortality for groups
(30-40 fish) of rainbow trout, Iron Gate Hatchery Fall Chinook and coho salmon exposed for 72 hours near
Beaver Creek in April, May, June, and September 2010 and then divided in half and held at either 13°C or 18°C
for 60 days. (coho were not exposed in April)
Comparison of percent loss from C. shasta for IGH fall Chinook and coho salmon exposed at 6 sentinel
sites in the Klamath River in June of 2007-2010 - High losses from C. shasta occurred in sentinel
Chinook and coho salmon held in the “infectious zone” i.e. near Beaver Creek and Seiad Valley during
2007-2009 (Figure 1.2.6, 1.2.7). No losses occurred in the upper Klamath River sites and very low
losses at Klamathon and Orleans in most years. In 2010, losses from C. shasta were lower than all
previous study years in the lower river.
5
FIGURE 1.2.6. Comparison of percent loss
with C. shasta infection of juvenile IGH fall
Chinook salmon exposed in the Klamath
River for 72 hr at six sentinel sites in June
2007- 2010.
FIGURE 1.2.7. Comparison of percent loss
with C. shasta infection of juvenile coho
salmon exposed in the Klamath River for 72
hr at five sentinel sites in June 2007- 2010
(coho were not held at Keno Eddy).
Comparison of sentinel results for the IGH Chinook and coho salmon exposed near Beaver Creek in
2007, 2008, 2009 and 2010 - From 2007 to 2009, there appeared to be a shift toward more severe
effects of C. shasta on the Chinook when compared to coho (Figure 1.2.8). In 2007 the loss of
juvenile coho was very high while the Chinook loss was lower. In 2008 both species suffered high loss
in May and June. In 2009, the greatest loss occurred in May and June in the fall Chinook. In general
however, losses for both species due to C. shasta have been high in May and June of 2007-2009. In
contrast, for 2010, cooler water temperatures in May appear to have greatly decreased the infection
and mortality from C. shasta for both the Chinook and coho during May and June.
6
FIGURE 1.2.8. Comparison of C. shastaderived mortality of juvenile IGH fall
Chinook and coho salmon exposed in the
Klamath River near Beaver Creek for 72 hr
in June 2007, 2008, 2009 and 2010.
Summary of the sentinel results Ceratomyxa shasta infections were detected in susceptible rainbow trout in all months tested
in 2010, i.e. April, May, June, and September near Beaver Creek and at all 6 sentinel sites
including the upper and lower Klamath River in May and June. Compared to previous year’s
sentinel results, C. shasta infections in IGH fall Chinook and coho were much lower in 2010.
Cooler water temperatures during exposures in May appear to have decreased the C. shasta
infection level in IGH Chinook and coho for both May and June. In May, only 2.6% of the
Chinook exposed at Seiad Valley and Keno Eddy died with C. shasta and none at 4 other sites.
In June, C. shasta infections occurred in the “infectious zone” of Beaver Creek and Seiad Valley
but the percent loss was lower than in previous years.

Juvenile Chinook and coho salmon became infected with C. shasta below Iron Gate Dam.
Highest mortalities followed exposure at the Beaver Creek site, then decreased downstream
to Seiad Valley and Orleans. This pattern has been consistent across all years (2007-2010) but
mortality was significantly lower in 2010. No infections occurred in the Chinook or coho near
Klamathon Bridge in 2010.

The lower Williamson River in the upper Klamath Basin was tested for C. shasta in May and
June. More than 90% of the rainbow trout died but no infections were detected in fall
Chinook or coho salmon.

Post-exposure water temperature affected percent mortality. No loss of Chinook and coho
salmon occurred at 13°C but low level losses (20% or less) were observed at 18°C.

Comparison of sentinel study results from 2007, 2008, 2009 and 2010 for fish exposed near
Beaver Creek shows a shift in level of mortality between coho and Chinook salmon. In 2007
coho loss in May and June was much higher than for Chinook. In 2008, both species suffered
very high losses after exposure. In 2009, the loss of Chinook was higher than coho from C.
shasta. In June 2010, losses in Chinook were again higher than coho i.e. 20% versus 10%, but
mortality for both species was much lower than in previous years.
7

No C. shasta infections were detected in susceptible rainbow trout exposed in May in the
lower Shasta River.
Task 1.3 WATER SAMPLE COLLECTION AND PARASITE DENSITY DETERMINATION
Water samples were taken to determine:
1. The spatial and temporal distribution of Ceratomyxa shasta in the Klamath River.
2. How abundance this year compares with levels in previous years.
3. If the distribution of the parasite has changed.
4. The relationship between parasite numbers measured in water samples and biological effects
in the different fish species.
Methods
Water samples were collected from 5 mainstem sites, Klamathon Bridge (KKB), Beaver Creek (KBC),
Seiad Valley (KSV), Orleans (KOR), and Tully Creek (KTC) once a week from March through September
in 2010, with an ISCO automatic sampler. The ISCOs were set for 24 h sampling beginning 8 am and 1
L was collected from the river every 2 hr for 24 hr, then the total sample was mixed manually and 4 x
1 L samples taken. Based on 2009 data, tributary sampling (Bogus Creek, Shasta, Scott and Salmon
Rivers) in 2010 was reduced to no sampling from May to September but increased in frequency at all
sites to weekly in March, April and October. Water was sampled manually (grab sample) at the
tributaries; 4 x 1 L were collected from the river at 1 time point. All samples were chilled until
filtered, within 24 hr of collection.
Water samples were also taken at the 6 sentinel fish sites during the exposures in May and June, and
only at Beaver Creek in April and September. Four 1 L samples were taken at the start and end of each
exposure. Samples were collected manually at the upper sites, WMR and KED, and primarily by ISCO
(supplemented by manual) at the lower index sites.
The modified DNA extraction procedure introduced in 2009, in which the filter paper was dissolved in
a series of acetone/ethanol washes prior to kit digestion, continued to be used in 2010. DNA was
extracted from 3 of the 4 frozen 1 L samples collected at each site. An inhibition test (IPC) was
performed on 1 sample from each site. Three samples per site were assessed with qPCR for the
presence of C. shasta DNA. Each sample was run in duplicate and sample pairs with values differing
by more than 1 Cq were rerun. Samples with 1 undetected well and 1 detected well of less than Cq
38 were rerun. Samples that were undetected (both wells) were assigned a Cq value of 42 and
included in the site average. Positive (tissue or artificial template) and negative (molecular grade
water) controls were included in each qPCR run. Each data point on a graph represents the average
of the 3 1L water samples collected at that time point; error bars display the standard deviation of
those 3 samples. Following the guidelines of Bustin et al. 2009, the term ‘quantification cycle’ (Cq) is
used for the cycle number at which a sample fluoresces and crosses a standard threshold.
Results & Discussion
C. shasta DNA was detected in water samples from all 5 lower Klamath River mainstem index sites
(Figure 1.3.1). At the uppermost site, Klamathon, less than 1 spore/L was present throughout the
collection period. Near Beaver Creek, density was above 1 spore/L in April, increased to above 10
spores/L in May and June, then decreased thereafter. A similar pattern and density was observed at
Seiad Valley. Levels were lower at Orleans, generally less than 1 spore/L, except on one occasion in
May and again in June. Levels at the most downstream site, Tully Creek, were already above 1
8
spore/L in April then increased to >10spores/L in May and 100 spores/L in June and July, then
decreased thereafter.
Klamathon
FIGURE 1.3.1. Density of Ceratomyxa shasta in 1L
water samples collected at five mainstem sites
from April through September in 2010. Each data
point is the average Cq of 3 x 1L water samples. A
lower Cq value indicates more parasite is present.
Cq levels equivalent to 1, 10 and 100 spores/L are
marked where appropriate. Sites are displayed in
a downstream direction: Klamathon, Near Beaver
Creek, Seiad Valley, Orleans, Tully Creek.
Near Beaver Creek
Orleans
Seiad Valley
Tully Creek
9
Only low densities of C. shasta were detected in the 4 tributaries assessed in March, April and
October. The highest level was 1 spore/L detected in the Scott River in October.
FIGURE 1.3.2. Density of Ceratomyxa
shasta in 1L water samples collected at four
tributary sites in March, April and October
in 2010. Each data point is the average Cq
of 3 x 1L water samples. A lower Cq value
indicates more parasite is present.
BOG=Bogus Creek, SCT=Scott River,
SHR=Shasta River, SMN=Salmon River.
With the exception of Tully Creek, spore densities were lower in 2010 than in 2009 (Figure 1.3.3,
1.3.4, 1.3.5). There was an order of magnitude difference in Spring; 1-10 spores/L were detected in
April of 2010 compared with 10-100 spores/L in 2009. Peak density levels were similar between the
two years, but occurred a month sooner in 2009. Traditionally, the highest levels of C. shasta are
detected at Beaver Creek and Seiad Valley, however, this year levels at Tully Creek surpassed these by
up to two orders of magnitude (in early summer).
FIGURE 1.3.3. Density of Ceratomyxa shasta in 1L water samples collected at five mainstem sites from April
through September in 2010 (upper graph) and 2009 (lower graph). Each data point is the average Cq of 3 x 1L
water samples. A lower Cq value indicates more parasite is present. Site abbreviations are explained in Table
1.1.1 above.
10
FIGURE 1.3.4. Comparison of
density of Ceratomyxa shasta
in 1L water samples collected
upstream of Beaver Creek
from April through September
in 2009 and 2010. Each data
point is the average Cq of 3 x
1L water samples. A lower Cq
value indicates more parasite is
present.
11
FIGURE 1.3.5.
Comparison of density of
Ceratomyxa shasta in 1L
water samples collected
at Tully Creek from April
through September in
2009 and 2010. Each
data point is the average
Cq of 3 x 1L water
samples. A lower Cq
value indicates more
parasite is present.
Comparison of spore densities in water samples collected at the beginning and end of the sentinel fish
exposures with mortality in trout and salmon revealed a different relationship this year. In previous
years, about 10 spores/L caused high mortality in Chinook. In 2010, despite similar parasite densities,
mortality was much lower. This suggests that another factor, likely lower temperatures in
Spring/early Summer in 2010, limited disease progression. In support, True et al. (2011) reported that
river flows below Iron Gate Dam were not substantially different in 2010 compared to previous study
years and they suggested that the cooler temperatures appeared to have played a more significant
role in disease dynamics in outmigrating juvenile Chinook salmon in 2010. Rainbow trout remained
the most sensitive species; when the parasite was detected in water samples, mortality occured in
sentinel fish.
1 spore/L
FIGURE 1.3.5. Sentinel water data
with corresponding sentinel fish data
(percent mortality due to Ceratomyxa
shasta) for April, May, June and
September 2010. In April, fish were
held only near Beaver Creek, and in
September only at Beaver Creek and
Seiad Valley. Each water data point is
the average of 3 x 1 L water samples
taken at the beginning and end of the
72 hour exposure. Rbt = rainbow
trout. A lower Cq value indicates
more parasite is present. WMR NC,
Williamson River Nature Conservatory;
KED, Keno Eddy; KKB, Klamathon
Bridge; KBC, near Beaver Creek; KSV,
Seiad Valley; KOR, Orleans.
12
MAY
JUNE
1 spore/L
SEPTEMBER
13
Summary of water sample data:





The temporal and spatial distribution of C. shasta differed in 2010 compared with 2009.
Levels were lower this year and peaked later.
The lowest peak spore densities, <1 spore/L, were detected at the most upstream site,
Klamathon Bridge.
The highest peak spore densities, >100 spores/L, were detected at the most downstream site,
Tully Creek.
Predominantly, low amounts of C. shasta DNA (<1 spore/L) were detected in the tributaries
(Bogus Creek, Shasta, Scott and Salmon Rivers).
In 2010, mortality was lower at similar parasite densities compared to previous years; 10
spores/liter did not cause high mortality in Klamath River salmon.
Task 1.4 POLYCHAETE ABUNDANCE AND INFECTION PREVALENCE
Samples of polychaete worms were collected to estimate:
1. M. speciosa density.
2. Prevalence of C. shasta infection in M. speciosa.
3. Any changes over time.
Methods
Polychaetes were sampled three times per year to coincide with sentinel fish exposures in May, June,
and September from 2007-2010. Samples were collected by targeting features that were considered
likely to support polychaetes (e.g., boulders in pools and eddies, Stocking et al. 2007). Polychaetes
were collected using a modified Hess sampler (a joint section of PVC pipe with an aperture 229 cm 2,
fitted with a 84µm collection net) and a scraping device. Samples were preserved in 70% ETOH in the
field and returned to the laboratory (J.L. Fryer Salmon Disease Laboratory, Oregon State University,
Corvallis, OR) for processing. To estimate field densities, the entire sample was placed into a sorting
tray (20cmx28cm, Wildco, FL) and three 25cmx25cm subsamples were randomly selected.
Subsamples were stained (20% Rose Bengal, Fisher Scientific) and polychaetes were counted using a
dissecting microscope (20-50x magnification). Density was calculated as mean number of polychaetes
in subsamples/area of subsample x area of sorting tray /total area of Hess (226.9cm2) and expressed
per m2.
Prevalence of C. shasta infection will be determined using the C. shasta-specific qPCR assay
developed in our lab (Hallett and Bartholomew 2006).
Results & Discussion
Polychaetes were found at all sites in all months, but overall polychaete density (averaged across all 3
months) was lowest at Seiad Valley (KSV) and highest at Tree of Heaven (TOH), although these
differences were not statistically significant due to large standard error values (Figure 1.4.1). At all
sites, polychaete densities were highest in June relative to other months (statistically significant at
KSV only). High levels of polychaete reproduction in late spring may increase drift and polychaete
movement into areas typically characterized by lower polychaete densities (e.g., Seiad Valley).
Examination of polychaete length frequency trends (in progress) may support this hypothesis.
Polychaete densities were higher (June and September sampling periods only) at TOH than other
index sites. This reach is characterized by increased food availability and relatively low water
velocities, which are hypothesized to be related to inputs from the Shasta River, whose confluence is
14
located between the I-5 and TOH index sites. The increased food in this reach may be diluted by
inputs from the Scott River, whose confluence is located between the TOH and KSV index sites. This
dilution, and the higher water velocities that result from the significant increase in water from the
Scott River, may explain why densities are generally lower at KSV. Examination of correlations
between polychaete densities and environmental variables (e.g., temperature and discharge) are in
progress.
FIGURE 1.4.1. Estimated polychaete densities at the three index sites in a) 2007, b) 2008, c) 2009, and d) 2010,
shown by sampling month at each index site. Values are means (+1 standard error). KSV=Seiad Valley,
TOH=Tree of Heaven, and I-5=I-5 Bridge. Note that in 2007 the I-5 and KSV sites were not sampled and TOH
was sampled only in June; in 2009 the KSV and I-5 sites were not sampled in May, and in 2010 the “June”
sampling period actually occurred from June 30-July 4.
Because our results, which are based on 3 subsamples per sample, were characterized by high
standard error values (over 55% of all samples fall into this group), we plan to process additional
subsamples to control for the possibility of laboratory processing bias before attributing the variability
among samples to natural field processes.
15
Task 1.5 PROJECT COORDINATION
In November, 2009, a meeting was held in Corvallis, OR with members of the OSU, USFWS and HSU
laboratories and Yurok tribe; a Karuk tribe representative joined us via conference call. Attendees
presented a summary of research conducted in 2009 and that planned for 2010. There was discussion
of protocols, outstanding research questions and data gaps and the 2007 management report was
revisited. A follow-up meeting was held at HSU with representatives from each research group in
March, 2010, immediately preceding the Klamath River Fish Health Workshop.
Objective 2. DEVELOPMENT OF A MODEL TO ADDRESS CRITICAL UNCERTAINTIES IN PARASITE
TRANSMISSION.
An epidemiological model depicting the life cycle of Ceratomyxa shasta was developed in 2009-10
(forming the core of Adam Ray’s Master’s thesis). This model identifies the parameters necessary for
propagation of the parasite and is being developed as a tool for identifying management strategies
that would be effective in improving the survival rate of out-migrating juvenile salmonids in the KR.
This model was published in Diseases of Aquatic Organisms (Ray et al. 2010) and a copy of that
manuscript has been appended to this report. In this study we quantified three parameters that were
identified from the model. The infectious dose of actinospores (A), the proportion of salmon infected
(S) and parasite induced mortality (π) were quantified by exposing Iron Gate Hatchery Chinook
salmon in the Klamath River. The quantification of these parameters led to the identification of a
non-linear mortality threshold for juvenile Chinook salmon to C. shasta ranging from 5.5 – 9.9 x 104
actinospores fish-1.
In 2010, we redesigned the epidemiological model to more thoroughly represent the complete life
cycle of Ceratomyxa shasta (Figure 2.1). This model now includes parameters pertaining to the
interactions between the parasite and the returning adult salmon, which are the most likely source of
myxospores in this system. Additionally, we have incorporated a developmental delay parameter
that will account for the time from when a host becomes infected to when the respective spore
stages are produced. This refinement of the model has led to 8 differential equations and the
realization additional information on the prevalence and intensity of infection in returning adult
salmon is necessary for further predictions.
16
FIGURE 2.1. Reconfiguration of the epidemiological model to include parameters addressing the interactions
between Ceratomyxa shasta and returning adult salmon.
Task 2.1. QUANTIFICATION OF TRANSMISSION RATE OF THE ACTINOSPORE TO THE SALMONID HOST
Methods
Three cages of 15 IGH Chinook were placed at 3 different velocities (0.01, 0.1, 0.2 m/s) in the Klamath
River at KBC. Cages were submerged for 3 hours and every 30 minutes water velocities were
measured at each cage and water samples were collected for analysis by qPCR. The measured
velocity and parasite density were used to estimate the challenge dose received by each cage. After
the 3 hour exposure, 5 fish from each cage were euthanized with an overdose of MS-222, and half of
the gill arch was removed and stored in 95% EtOH for qPCR analysis. The remaining fish were
transferred to the SDL and reared to determine any parasite-associated mortality. qPCR gill standards
were achieved by placing 1 and 8 actinospores onto uninfected gill and processing. These standard
values allowed for the estimation of the number of actinospores that attached to the gill at the
different velocities.
17
Results & Discussion
Transmission data was analyzed in two ways: 1) by determining the transmission efficiency of the
actinospore as a ratio of attached versus total dose and 2) by determining the total number of
actinospores attached. Using the first analysis, the transmission efficiency appears to decrease as
velocity increases (Figure 2.1.1). In contrast, comparison of the total number of actinospores
attached showed no difference between the velocities, suggesting a consistent attachment rate
regardless of velocity (Figure 2.1.2). These conflicting results suggest that the decreased transmission
efficiency may be a result of a greater exposure dose and not from a decreased ability of the
actinospore to attach. This has led to design of two controlled laboratory experiments that examine
the effects of different parameters on attachment. The first is an extension of the velocity
experiment, using a swim tube to obtain greater velocities and determine if there is a velocity related
threshold above which actinospores can no longer attach. The second is a temperature study to
determine if water temperatures affect the actinospore attachment rate. As the parasite enters
through the gills, the number of times a fish operculates (vents) its gill is likely to affect parasite
attachment. Operculation and respiration increase with temperature, thus at a higher temperature
fish there is increased potential for actinospore transmission. Data on how these two environmental
parameters affect transmission will provide a range of values and conditions that can be incorporated
into the epidemiological model.
Transmission efficency
1.0E+00
1.0E-01
FIGURE 2.2.1. Transmission
efficiency of Ceratomyxa
shasta actinospores to Iron
Gate Hatchery Chinook
salmon exposed in the
Klamath River near Beaver
Creek for 3 hours at 3
different velocities (0.02,
0.1, 0.2 m/sec).
1.0E-02
1.0E-03
1.0E-04
1.0E-05
1.0E-06
0
0.05
0.1
0.15
0.2
0.25
Velocity (m/sec)
18
100
Slow
Medium
Fast
Total C. shasta attached
90
80
70
60
50
40
30
20
FIGURE 2.2.2. Total number
of Ceratomyxa shasta
attached to the gills of Iron
Gate Hatchery Chinook
salmon held at 3 different
velocities (0.02, 0.1, and 0.2
m/sec). There is no
difference among the 3
velocities in the number of
parasites attached.
10
0
Task 2.2. QUANTIFICATION OF TRANSMISSION RATE OF THE MYXOSPORE TO THE POLYCHAETE HOST
Two laboratory experiments were conducted to examine the transmission of the myxospore to the
polychaete (η2) and to measure the resulting prevalence of infection in the polychaete host (P). A
known quantity of myxospores was added to polychaetes in astatic system and the polychaete
population was assayed by qPCR to determine the infection prevalence. Polychaete survival was
satisfactory in the first experiment but transmission was low and in the second experiment survival
was poor. The limited data collected will be incorporated into the epidemiological model; however
future data for this parameter may be limited to coarse approximations from field surveys of
polychaete populations throughout the Klamath River.
Task 2.3. MODEL VALIDATION AND DETERMINING INFORMATION GAPS
Model validation and data synthesis are ongoing; however we have identified data gaps
corresponding to returning adult salmon and parasite interaction parameters.
Objective 3. DEVELOPMENT OF A QPCR ASSAY TO DIFFERENTIATE BETWEEN THE DIFFERENT ITS1 STRAINS
OF CERATOMYXA SHASTA.
Task 3.1. RE-ASSESSMENT OF STANDARD QPCR ASSAY.
We use a DNA-based assay (quantitative PCR) to detect and quantify Ceratomyxa shasta in water
samples. The assay was developed to be specific for our target parasite, based on available data.
However, in 2009, a new species of Ceratomyxa was discovered in the Klamath River, in sticklebacks.
The stickleback parasite differs morphologically and genetically (ssu rRNA 96% similar) to C. shasta
but none of the base differences are in the 18S annealing locations targeted by the qPCR primers and
probe (Figure 3.1.1) and thus it is amplified in the standard qPCR assay. There is a two-base insertion
between the probe and reverse primer, which renders the stickleback Ceratomyxa longer than C.
shasta.
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FIGURE 3.1.1. Sequence alignment of Ceratomyxa shasta and the Ceratomyxa sp. from sticklebacks showing
the annealing locations of the qPCR primers and probe and the two-base insertion between the probe and
reverse primer. Both species can be amplified in the C. shasta qPCR but may be distinguished with melting
curve analysis.
The stickleback Ceratomyxa is also amplified by the C. shasta ITS genotyping primers but there is
much variation in the ITS region and its sequence can be readily distinguished from C. shasta as a
different genotype (Figure 3.1.2). To determine whether the novel Ceratomyxa contributes to the
spore densities (Cq values) currently attributed solely to C. shasta, we sequenced the ITS region
(standard genotyping assay) of a range of water samples from different sites (including Tully Creek,
our closest monitoring site to the known location of infected sticklebacks) collected at different times
of the year and scrutinized them for the novel Ceratomyxa which would appear as a different
genotype.
FIGURE 3.1.2. Sequence alignment of part of the ITS gene of Ceratomyxa shasta and the Ceratomyxa sp. from
sticklebacks showing the three-base ATC repeat characteristic of C. shasta genotype III and the variation
between the two species.
All genotypes detected in the sequencing reaction were attributable to C. shasta; there was no
evidence of the stickleback Ceratomyxa. The detection limit of the genotyping assay is about 5%, thus
if the stickleback Ceratomyxa was present it was at very low levels.
Unique sequences, such as those of C. shasta and the stickleback Ceratomyxa, may be differentiated
with high resolution melting curve analysis (HRM). We plan to amplify extracted DNA from water
samples using the standard qPCR primers in a qPCR but with a saturating dye included instead of the
probe. The dye will bind to any amplified product (double-stranded DNA) and fluoresce when the dsDNA is melted. A melting curve will be produced that is characteristic for each unique sequence
present. We will prepare reference samples with known combinations of the two Ceratomyxa
species, quantify the DNA present and assess whether the HRM can distinguish between them. We
will then assess a range of water samples from different sites collected at different times of the year.
With the assistance of the Yurok Tribe and the USFWS CA-NV FHC we plan to conduct more extensive
sampling of stickleback fish in the Klamath River to determine the overlap in range of the two
Ceratomyxa parasites.
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Task 3.2. VALIDATION OF NEW QPCR/HRM ASSAY TO SCREEN SAMPLES FOR ITS GENOTYPES
Our DNA sequencing studies of the ITS1gene of Ceratomyxa shasta revealed 4 different strains (ITS
genotypes) of the parasite in the Klamath Basin which differentially cause mortality in different
salmonid hosts e.g. type I is pathogenic for Chinook salmon whereas type II is detrimental to coho.
PCR followed by sequencing remains the best method to distinguish between C. shasta ITS genotypes
but this approach does not quantify the various genotypes concurrently present in a sample and does
not favor high throughput sample processing. In 2009, we took advantage of equipment newly
acquired by the university and developed a promising novel molecular assay that combined qPCR with
high resolution melting curve analysis (HRM). The approach was less laborious and less expensive
than traditional sequencing, however, it was not useful for distinguishing genotypes in mixed
samples, nor could it quantify the relative abundance of each genotype in a sample. In 2010, we
explored another molecular method, pyrosequencing, which had the potential to detect and quantify
all genotypes present, including novel ones. We worked with the molecular company technicians to
develop appropriate software, but the ITS1 gene proved unsuitable because it was too variable. We
then targeted a second gene, which codes for the heat shock protein, with the hope that it would
mirror the division of ITS1 genotypes but exhibit less variation. We designed primers and developed
an assay and sequenced multiple samples of each ITS1 genotype. Unfortunately, the HSP gene did
not resolve the ITS1 genotypes and we did not pursue it for pyrosequencing.
The ability to detect and measure the relative abundance of the strains specific for Chinook and coho
salmon will be important for monitoring the effects of methods implemented for reducing C. shasta
levels in the lower basin. We plan to continue to explore candidate genes suitable for distinguishing
and quantifying the various C. shasta strains.
Task 3.3. SYNTHESIS OF MULTI-YEAR WATER SAMPLE DATA
We have multiple years of water sample data from the Klamath River (2006-20010). qPCR
methodology varied from year to year (e.g. change of qPCR machine, introduction of protocol for
dissolution of filter membrane) and qPCR water sample data has now been standardized to allow
multi-year comparisons. Data spanning multiple documents have been combined. The data are being
scrutinized for correlations between salmonid mortality and temperature and also mortality and
parasite density in water samples. Two manuscripts examining these relationships are in preparation.
These data have also been synthesized with data from the USFWS CA-NV FHC and the USGS Columbia
River Research Laboratory for inclusion in a Conceptual Model for Disease Effects in the Klamath
River.
___________________________________________________________________________________
ACKNOWLEDGEMENTS
This report was prepared by: Sascha Hallett, Rich Holt, Adam Ray, Julie Alexander, Gerri Buckles, and
Stephen Atkinson. The Karuk and Yurok tribes assisted with water sample collection. Stephen
Christy, Taylor Derlacki, and Kourtney Hall (OSU) assisted with qPCR (water samples). Jill Pridgeon
(OSU) helped filter water samples.
We thank: California Department of Fish and Game, especially Kim Rushton and crew at the Iron Gate
Hatchery for providing Klamath River fall Chinook and coho salmon juveniles for our sentinel studies;
Roaring River Hatchery, Oregon Department of Fish and Wildlife, Scio, OR for susceptible rainbow
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trout; Scott Vanderkooi and crew USGS, Klamath Falls, OR who assisted with sentinel cage studies at
Keno Eddy, Keno, OR.
We are grateful to the land owners who allowed us access to conduct the sentinel studies and water
sampling:
The Nature Conservancy, Klamath Falls, OR for access at the lower Williamson River;
The Sportsman’s Park Club near Keno, OR;
Klamath River Country Estates near Klamathon, CA;
Fisher’s RV Park at Klamath River, CA;
Wally Johnson, Seiad Valley;
Sandy Bar Resort, Orleans, CA.
REFERENCES
Hallett, S. L. and J. L. Bartholomew. 2006. Application of a real-time PCR assay to detect and quantify the
myxozoan parasite Ceratomyxa shasta in water samples. Diseases of Aquatic Organisms 71:109-118.
Hallett, S. L. and J. L. Bartholomew. 2009. Development and application of a duplex QPCR for river water
samples to monitor the myxozoan parasite Parvicapsula minibicornis. Diseases of Aquatic Organisms 86:3950.
Ray, A.R., P. A. Rossignol and J. L. Bartholomew. 2010. Mortality threshold for juvenile Chinook salmon
Oncorhynchus tshawytscha in an epidemiological model of Ceratomyxa shasta. Diseases of Aquatic
Organisms 93:63-70.
Stocking, R. W. and J. L. Bartholomew. 2007. Distribution and habitat characteristics of Manayunkia
speciosa and infection prevalence with the parasite, Ceratomyxa shasta, in the Klamath River, OR-CA, USA.
Journal of Parasitology 93:78-88.
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