An Examination of Environmental Factors Associated with Myxobolus ...

Journal of Aquatic Animal Health 18:90–100, 2006 Copyright by the American Fisheries Society 2006 DOI: 10.1577/H05-017.1

[Article]

An Examination of Environmental Factors Associated with Myxobolus cerebralis Infection of Wild Trout in Pennsylvania ADAM J. KAESER*1 Pennsylvania State University, University Park, Pennsylvania 16802, USA

CHARLOTTE RASMUSSEN U.S. Geological Survey, Western Fisheries Research Center, Seattle, Washington 98115, USA

WILLIAM E. SHARPE Pennsylvania State University, University Park, Pennsylvania 16802, USA Abstract.—Salmonid whirling disease, caused by the myxosporean parasite Myxobolus cerebralis, was first observed in the United States in 1956 in central Pennsylvania. The parasite was subsequently discovered at several culture facilities throughout the state, and widespread distribution of this parasite via the stocking of subclinically infected brook trout Salvelinus fontinalis, rainbow trout Oncorhynchus mykiss, and brown trout Salmo trutta has been assumed. Although no monitoring of wild populations occurred until the late 1970s, it is a common belief that epizootics of whirling disease, now realized in the Intermountain West, are unlikely to have occurred in Pennsylvania. We conducted a review of historical information and a synoptic survey aimed at identifying factors that may prevent whirling disease outbreak in this region, reasoning that such information might be useful in identifying management strategies for populations affected by this parasite. Here we present data on parasite prevalence, fish populations, stream attributes, and the genetics of Tubifex tubifex (the obligate oligochaete host for the parasite) to evaluate various hypotheses proposed for low whirling disease impact in the region. We did not find clear associations between factors such as stream gradient, the genetics of T. tubifex populations, or the composition of resident trout populations and the pattern of M. cerebralis occurrence in Pennsylvania. We suggest that this pattern may be best explained by the association between T. tubifex host populations and point sources of organic enrichment. The potential restriction of T. tubifex populations to locations near sources of organic enrichment may be a factor in explaining why whirling disease has not been observed to cause population declines among wild trout in this region and should be further investigated.

Whirling disease was first observed in the United States in 1956 among brook trout Salvelinus fontinalis at the Benner Spring Fish Culture Station in Centre County, Pennsylvania (Hoffman 1962). For decades, hatchery-reared brook trout, rainbow trout Oncorhynchus mykiss, and brown trout Salmo trutta infected with Myxobolus cerebralis were released among wild populations (Bartholomew and Reno 2002), leading fisheries professionals to speculate that the parasite had been established throughout the state of Pennsylvania. In the absence of conspicuous declines of wild trout populations, a belief gradually emerged that whirling disease posed a threat only to trout reared in hatcheries.

The emergence of whirling disease among wild trout populations in Colorado and Montana in the early 1990s dramatically overturned this belief (Nehring and Walker 1996; Vincent 1996). Detection of the parasite throughout the Intermountain West stimulated a massive research effort to understand and combat the pathogen. Although several questions regarding whirling disease have been resolved, explaining local and regional disparities in disease outbreak remains a focus of ecological research (Downing et al. 2002; Kerans et al. 2004). Understanding the dynamics of M. cerebralis has important, practical management applications given that the elimination of this parasite from wild trout populations appears unlikely (Wagner 2002). Moreover, the parasite continues to invade new and remote territory in the western United States despite the cessation of stocking of infected trout. Several species of salmonids are susceptible to whirling disease, including the eastern brook trout (Thompson et al. 1999; Vincent 2002), raising questions regarding past

* Corresponding author: [email protected] 1 Present address: Georgia Department of Natural Resources, Wildlife Resources Division, Fisheries Management, 2024 Newton Road, Albany, Georgia 31701-3576, USA. Received March 22, 2005; accepted November 2, 2004 Published online April 25, 2006

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or future impacts of this parasite in other regions including the eastern United States and Canada. Much less is known about M. cerebralis in regions outside the Intermountain West. If epizootics have not occurred among wild trout populations in other regions where M. cerebralis has been introduced, then searching for ecological differences at a broader scale through comparative field research may provide insight into the conditions necessary for disease outbreak. A lengthy history of M. cerebralis in Pennsylvania makes this state a logical choice for such an investigation. Our overall objective was to identify the most likely factor(s) contributing to the low whirling disease impact in Pennsylvania, and to identify M. cerebralis-positive systems for further investigation. Therefore, we conducted a review of historical data and performed a synoptic survey to evaluate associations between parasite occurrence and various disease risk factors (Kerans and Zale 2002). Higher infection risk has been associated with the following factors: lower stream gradients or elevation (Schisler and Bergersen 2002); proximity to point sources of organic enrichment where high densities of the aquatic oligochaete Tubifex tubifex (the obligate second host for the parasite) are commonly observed (Zendt and Bergersen 2000; Allen and Bergersen 2002; Thompson et al. 2002); the predominance of genetic lineages of T. tubifex susceptible to infection by the parasite (Beauchamp et al. 2002, 2005; Kerans et al. 2004); the spatiotemporal overlap of susceptible juvenile stages of trout and the release of infective, triactinomyxon spores of M. cerebralis with respect to water temperature (ElMatbouli et al. 1999; Downing et al. 2002; MacConnell and Vincent 2002; Ryce et al. 2004); and high stream conductivity (Sandell et al. 2001). We examined the following factors: (1) composition and abundance of the resident trout population, (2) stream gradient, (3) the existence of point sources of organic enrichment, (4) the susceptibility of T. tubifex as assessed by genetic markers, (5) stream temperature with respect to the appearance of juvenile trout stages and the potential for high concentrations of triactinomyxon spores, and (6) specific conductance. The distribution and abundance of aquatic oligochaetes was examined in Kaeser and Sharpe (2006, this issue). Methods Historical data.—We examined a comprehensive data set from the Pennsylvania Fish and Boat Commission (PAFBC) containing the results of any screenings for M. cerebralis among wild or hatcheryreared trout stocks between 1956 and 1999 for patterns in the historical occurrence of M. cerebralis among wild trout. Much of the historic sampling occurred during 1977, the year the PAFBC first investigated the

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establishment of the parasite among wild trout populations in streams that had been stocked with trout from M. cerebralis–positive fish culture facilities (O’Grodnick 1978). Fish were analyzed by the PAFBC using either the plankton centrifuge (O’Grodnick 1975) or the pepsin–trypsin digest (PTD) method for extraction and examination of parasite myxospores (the hardened spore that infects T. tubifex hosts) following standard protocols (Thoesen 2004). In most cases testing involved pooled fish samples (five-fish pools). We calculated maximum likelihood estimates of apparent prevalence and associated confidence intervals from these data using a SAS program developed for this purpose (SAS Institute 1995; Williams and Moffitt 2001). Using the number of fish examined in each case, we calculated the minimum expected prevalence of M. cerebralis that would be detectable at a 95% level of confidence using the formulae and program FreeCalc, version 2, developed by Cameron and Baldock (1998a, 1998b). In this procedure we assumed a local population size of 3,000 trout, an assumption that yields conservative estimates of minimum expected prevalence, an assay specificity of 1.0, and a sensitivity of 0.83 for the PTD method (Markiw and Wolf 1974). For all streams sampled, we derived an estimate of stream gradient as the change in elevation over stream channel length using topographic maps provided by the software Toposcout, version 2.01 (Maptech, Inc., Greenland, New Hampshire). For small streams (first or second order), the gradient was estimated from the upstream point at which the stream was defined as perennial on the map, to the downstream mouth of the stream. For larger streams the gradient was determined over the entire length of stream within that order. For example, if a third order stream site had been sampled, then we determined the gradient for the entire third order segment of the stream. We reviewed PAFBC records to obtain trout population data, obtained as close in space and time to the M. cerebralis screening events as possible, to prospect for relationships between parasite occurrence and trout population composition and abundance. Mark–recapture methods were primarily used during PAFBC population surveys. Species density was estimated using the Chapman modification of the Petersen formula (Chapman 1951) and the average width and length of the study reach. Survey site selection.—The PAFBC data set guided our selection of lower-gradient sites that were located in watersheds that had either been previously screened for M. cerebralis or that contained wild populations of rainbow trout Oncorhynchus mykiss to target in our survey. Sites from the Spring and Big Fishing Creek watersheds of Centre and Clinton counties were

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chosen, as were three streams in south-central Pennsylvania (Kaeser and Sharpe 2006, this issue). One additional low-gradient stream, Piney Creek, was later added to the survey. Higher-gradient stream sites that were known to contain sympatric populations of rainbow trout and brook trout were identified for the survey. Several sites meeting these criteria were located in the Loyalhanna Creek watershed in Westmoreland County: Wildcat Run, Fowlers Run, McGinnis Run, Powdermill Run, Laughlintown Run, and Furnace Run. Fortuitously, we were aware of an outbreak of whirling disease that had occurred in 1996 at a small culture facility in this watershed and were granted access by landowners to work at the nearby sites of interest. One additional high-gradient site, Tubmill Run, was included in the survey on the basis that it contained brook trout and rainbow trout. Our survey encompassed roughly one-third of all streams known to contain wild rainbow trout populations in Pennsylvania. Fish collection and analyses.—During the summers of 2001–2003, we visited stream sites to collect fish population data. At each site we selected a 100-m reach that contained several areas of fine sediment deposition. The sediment deposits were important to reach selection because on separate occasions, we sampled these areas for aquatic oligochaetes (Kaeser and Sharpe 2006). We conducted three-pass, backpack electrofishing surveys of trout populations in each 100m reach. We examined all fish for the presence of clinical signs of whirling disease (e.g., blacktail, skeletal deformities), and weighed and measured individuals. Population estimates were calculated using the program MicroFish (Van Deventer and Platts 1985). During these surveys we removed a sample of 15 age-1 or older wild trout from each site or from areas immediately upstream if necessary. The maximum number of trout we were permitted to obtain was 15 per site. Age-1 wild trout were distinguished from age-0 and older wild fish and hatchery fish by size and physical condition. We collected rainbow trout whenever available; otherwise, samples of brook trout or brown trout Salmo trutta were taken. Fish samples collected during the 2001–2003 surveys, with the exception of McGinnis Run fish, were screened for infection by M. cerebralis by the U.S. Fish and Wildlife Service’s Northeast Fishery Center in Lamar, Pennsylvania. Half-heads from each fish were examined using the pepsin–trypsin digest method following standard protocols (Thoesen 2004). Digested tissue preparations were examined under light microscopy for the presence of mature spores of the parasite. If myxospores, or spore-like particles, were observed in a half-head sample, the remaining halfhead was screened for M. cerebralis DNA using polymerase chain reaction (PCR) methods (Andree et

al. 1998). We interpreted a positive PCR result as confirmation of infection by M. cerebralis. McGinnis Run trout were examined for infection by M. cerebralis during an independent fish health study conducted by the Conservation Fund, Shepherdstown, West Virginia. Cranial histologic sections from these fish were prepared by personnel at the Conservation Fund, and then examined microscopically by Ana Baya, fish health inspector at the Animal Health Laboratory of the Maryland Department of Agriculture, College Park, for lesions and the presence of M. cerebralis myxospores. Following this inspection, we procured the samples and sent only those exhibiting signs of infection to the Washington Animal Disease Diagnostic Laboratory at Washington State University, Pullman, Washington, to conduct a second microscopic examination. Here we report the more conservative of the two estimates of infection prevalence among the McGinnis Run trout. Temperature and water quality measurements.— Temperature and specific conductance were recorded using handheld devices during visits to the study sites in 2001–2003. A grab sample of stream water was obtained in a clean polyethylene bottle during a summer visit at each site, transported on ice, and submitted to the Penn State Institutes of the Environment Water Laboratory, University Park, Pennsylvania, for pH and acid neutralizing capacity (ANC) analyses. In January 2003, we installed Tidbit temperature monitors (Onset Computer Corporation, Bourne, Massachusetts) at the 10 most accessible study sites. Six low-gradient stream sites (Spring Creek upper, Spring Creek lower, Logan Branch, Trindle Spring Run, Big Spring Creek, and Falling Springs Branch) and four high-gradient stream sites (Big Fishing upper, Galbraith Gap, Wildcat Run, and McGinnis Run) were selected. The temperature monitors recorded stream temperature at 30-min intervals and were maintained for 1 year. Mean daily temperatures were calculated for each record. T. tubifex genetics.—We collected worms at the following sites for genetic analyses: Cedar Run (Clinton County), Cedar Run (Centre County), Piney Creek, an unnamed tributary to Falling Springs Branch that drained a fish culture facility, and a small rearing pond that drained into McGinnis Run. This list comprised the majority of sites that could provide sufficient numbers of mature T. tubifex for the analysis. A total of 60 worms were assayed from each site, with the exception of the Falling Springs Branch tributary where we obtained 48 specimens. Mature or otherwise large worms were extracted from mud collected at the sites and examined under a stereodissection scope to confirm the presence of hair chaetae, and the anterior portion of each worm was fixed in formalin and slide mounted for species identification following Kathman


and Brinkhurst (1998) prior to genetic analysis. Genomic DNA was isolated from the posterior portion of each worm using the Nucleospin Multi-96 Tissue Kits as per manufacturer’s protocol (Clontech Laboratories, Inc., Palo Alto, California). The lineage of T. tubifex individuals from each stream was determined using the mitochondrial 16S gene primers developed by Beauchamp et al. (2002). Polymerase chain reactions contained 20 pmol of each primer (I, III, V, VI, and tub16SR), 200 lM deoxynucleotide triphosphates, 2.5 mM MgCl2, one unit of Taq polymerase (Promega Corp., Madison, Wisconsin), 13 Rediload (Research Genetics, Inc., Huntsville, Alabama), 10 mM tris-HCl (pH 9.0 at 258C), 50 mM KCl, and 0.1% Triton X-100. Polymerase chain reaction amplification was performed on a PTC-100 thermocycler (MJ Research, Inc., Watertown, Massachusetts). After an initial denaturation step of 958C for 5 min, amplification was achieved by 35 cycles of a 958C denaturing step for 1 min followed by primer annealing at 628C for 1 min, and extension at 728C for 1 min. Polymerase chain reaction products were visualized by agarose gel electrophoresis on 2.25%, 0.5 3 TAE gels. To accurately assess mtDNA lineages, amplification results were compared with those of two T. tubifex individuals from each mtDNA lineage (I, III, V, and VI) that were amplified and visualized on agarose gels along with the unknown stream samples. Data analysis.—A statistical analysis of the factors associated with M. cerebralis occurrence or prevalence among fish samples was not conducted due to the following limitations in the data set: the samples were composed of one or more different species and ages of fish that reflected varying levels of susceptibility and exposure to infection by M. cerebralis (MacConnell and Vincent 2002), and some samples were obtained at sites within the same drainage or at the same site during multiple years and thus could not be considered independent. Therefore, qualitative interpretations were deemed more appropriate. Results M. cerebralis Occurrence Historically, the highest values of apparent prevalence (20–77% of fish infected; n ¼ 13–140) were observed during Pennsylvania Fish and Boat Commission surveys conducted at Big Spring Creek, Big Fishing Creek, Buffalo Run, and Falling Springs Branch (Table 1). The parasite was detected in multiple years in Big Fishing Creek, Falling Springs Branch, and Cedar Run in Clinton County (see also O’Grodnick 1979). Pennsylvania Fish and Boat Commission records indicated that M. cerebralis was detected among captive trout at the Tylersville fish culture

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facility discharging to Big Fishing Creek during 15 of the last 18 years of screening. Long-term monitoring of the resident trout population at a site located 1.6 km below this facility indicated that a trout population composed of brown trout and brook trout has inhabited the reach during the last two decades. Population surveys on Falling Springs Branch indicated that an abundant wild rainbow trout population has inhabited this stream for several decades (Figure 1). During the 2001–2003 survey, we examined a total of 871 trout among the survey sites. None of these fish exhibited clinical signs of whirling disease. Trout infected with M. cerebralis were found at 4 of the 19 sites sampled: McGinnis Run, Falling Springs Branch, Piney Creek, and Cedar Run in Clinton County. Prevalence was highest among brook trout from McGinnis Run (35% infected; n ¼ 20). Due to small sample sizes, we were unlikely to detect infection, if it existed, at levels of prevalence less than 22%. Of the 20 cases where M. cerebralis was detected in Pennsylvania over the last 25 years, 16 detections occurred at sites located along a stream that received discharge from a fish culture facility. The remaining four cases of detection occurred at sites that were either tributaries to a stream receiving discharge from a fish culture facility (Cherry and Roaring runs), or were sites situated in a landscape dominated by agricultural practices (Piney Creek and Buffalo Run; Kaeser 2004). Big Fishing Creek and Buffalo Run were among those streams sampled in 1977 that had been stocked with trout that originated from M. cerebralispositive fish culture facilities. The stocking history of Piney Creek was not known. Lowland, spring-fed stream sites typically exhibited topographic stream gradients of 0.8% or less, whereas higher-elevation mountain streams exhibited gradients of 1.0% or more (Table 1). M. cerebralis was detected among trout residing in both high-gradient (e.g., McGinnis Run) and low-gradient (e.g., Falling Spring Branch) stream sites. In Kaeser and Sharpe (2006) we used a digital elevation model to estimate slope for the 2001–2003 survey sites. The two alternative measures of stream gradient were linearly related (% W-slope ¼ 1.43 3 [stream gradient %] þ 0.85; r2 ¼ 0.81; P , 0.001). M. cerebralis infection was detected among lowand high-density populations of trout and among mixed-species assemblages (Table 2). For example, the parasite was detected among a depauperate trout population (;200 trout/ha) composed solely of brown trout (Cedar Run in Clinton County) and at Falling Spring Branch, where the most abundant rainbow trout population (;2,500–3,500 trout/ha) in a Pennsylvania stream is located.

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TABLE 1.—Prevalence of Myxobolus cerebralis among wild trout from Pennsylvania stream sites (1974–2003). Our recent survey data are indicated by bold italics to distinguish them from Pennsylvania Fish and Boat Commission data.

Stream site Big Fishing Creek (upper) Big Fishing Creek (mid)f Big Fishing Creek (upper) Big Fishing Creek (mid) Big Fishing Creek (mid) Big Fishing Creek Big Fishing Creek (mid) Big Fishing Creek (mid) Cedar Run Elk Creek (lower) Elk Creek (upper) Spring Creek Spring Creek Spring Creek Spring Creek Logan Branch Falling Springs Branch Falling Springs Branch Falling Springs Branch Falling Springs Branch Big Spring Creek Big Spring Creek Big Spring Creek McGinnis Run Buffalo Run Cherry Run Roaring Run Roaring Run Spanglers Gap Run McCaleb Run Little Fishing Creek Rauchtown Creek Rauchtown Creek Cedar Run Galbraith Gap Run Benner Run Big Poe Creek Marsh Creek Standing Stone Wallace Run Wallace Run Porcupine Run Porcupine Run Reese Run Laughlintown Run Furnace Run Powdermill Run Tubmill Run Wildcat Run Fowlers Run Piney Creek Trindle Spring Run Trindle Spring Run a

County f

CLN CLN CLN CLN CLN CLN CLN CLN CLN CEN CEN CEN CEN CEN CEN CEN FRK FRK FRK FRK CMB CMB CMB WES CEN CLN CEN CEN CLN CLN CLN CLN CLN CEN CEN CEN CEN CEN CEN CEN CEN VEN VEN VEN WES WES WES WES WES WES BLR CMB CMB

a

Year

Sample size

Species testedb

Apparent prevalencec

1977 1977 1984 1984 1994 1997 2001 2002 2003 1977 1977 1974 1994 2002 2002 2002 1983 1995 1997 2001 1977 1978 2002 2001 1977 1977 1977 1984 1977 1977 1977 1977 1997 2002 2002 1977 1977 1977 1977 1977 1977 1977 1997 1977 2001 2001 2001 2001 2001 2002 2003 1997 2001

115 50 30 25 40 30 15 15 15 50 60 6 20 15 15 15 34 30 30 15 25 13 13 20 140 60 55 12 60 30 60 15 15 10 15 120 105 60 115 48 42 29 30 30 15 15 15 15 15 14 15 30 15

Brk Brk Brk Brk Brk brn Brk Brk Brn Brn Brk, brn Brk Rbw Brn Brn Brn Rbw, brn Rbw Rbw Rbw Rbw Brk, rbw Brk, brn Rbw Rbw, tgr, brk, brn Brn Brk Brk Brk Brk Brk Brk Brk Brk Brn Brk, brn Brk Brk, brn Brk Brk, brn Brk Brk, brn Brk, rbw Rbw Rbw Rbw Rbw Rbw Rbw Brk, rbw Brk Brn Rbw Rbw

26 All þg 10 10 13 33

95% confidence interval

Minimum detectable prevalenced

16–39

4 7 14 14 9 12 22 22 22 7 6 50 17 22 22 22 3 12 12 22 14 25 25 17 3 6 6 27 6 12 6 22 22 32 22 3 4 6 3 8 9 12 12 12 22 22 22 22 22 24 22 12 22

2–28 2–28 4–28 16–50

7 13

0–19 5–26

All þg 24

6–56

29 30

14–45 11–60

13 28 77

0–31 9–58 54–100

35 32 4 4

14–56 21–46 1–11 1–11

7

0–19

Gradient (%) 0.18 1.01 0.18 1.01 1.01 1.01 1.01 0.59 0.41 2.39 0.23 0.23 0.23 0.49 0.79 0.42 0.42 0.42 0.42 0.34 0.34 0.34 2.29 0.55 1.20 1.06 1.06 4.75

Fish culture facilitye Y Y Y Y Y Y Y Y Y Yh Y Y Y Y Y Y Y Y Y Y Y Yh Y

Y Y

1.61 2.56 2.56 0.53 2.07 1.92 1.58 1.24 1.72 1.26 1.96 1.33 1.33 1.36 2.52 5.20 3.61 4.75 6.26 3.29 0.68 0.21 0.21

County codes are as follows: CLN ¼ Clinton, CEN ¼ Centre, FRK ¼ Franklin, CMB ¼ Cumberland, WES ¼ Westmoreland, VEN ¼ Venango, and BLR ¼ Blair. b Samples included the following species: brk ¼ brook trout, rbw ¼ rainbow trout, brn ¼ brown trout, and tgr ¼ tiger trout (brown trout 3 brook trout). c Calculated using the formulae of Williams and Moffitt (2001) for pooled samples; otherwise prevalence values were reported as the number infected/total and the 95% confidence intervals were calculated from the exact binomial distribution. In all samples obtained prior to 2001, infection was assessed using the pepsin–trypsin digest (PTD) method. Samples obtained during 2001–2003 were first examined by PTD, and those found to be infected were then subjected to polymerase chain reaction to confirm the presence of M. cerebralis DNA. Fish sampled from McGinnis Run were subjected only to histological examination of infection by M. cerebralis. Blank species in this column indicate that the parasite was not detected. d Calculated from the formulae of Cameron and Baldock (1988a, 1988b) at a 95% confidence level.


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and Cedar Run (Clinton County), we found both abundant T. tubifex worms and infected brown trout. T. tubifex lineages I and III were predominant and co-occurring at all five sites (Figure 2). A few worms belonging to lineage VI were found at Cedar Run in Clinton County. Stream Temperature and pH

FIGURE 1.—Trends in the abundance of rainbow trout, brown trout, and brook trout in Falling Springs Branch, Franklin County, Pennsylvania, during the period 1984–2001. Points represent the mean and error bars the range for multiple population estimates during a given year. Estimates were obtained at three sites spanning 1.9 km of stream channel, except in 1987 and 2001.

Tubifex tubifex Associations T. tubifex worms were found at 10 of the 20 sites included in the 2001–2003 oligochaete distribution survey. The occurrence and abundance of T. tubifex was related to linear distance to suspected upstream organic sources of enrichment (Kaeser and Sharpe 2006). Among sites where T. tubifex was found, the relative abundance and density of this species was generally low, with the exception of the Cedar Run (Centre County) and upper Big Fishing Creek sites. Both of these sites exhibited high abundances of T. tubifex worms. The Cedar Run site was located adjacent to a riparian cattle pasture, and the Big Fishing Creek site was located just downstream of the Tylersville fish culture facility that was notably discharging effluent high in organic matter. The site-specific abundance of T. tubifex worms appeared unrelated to the detection of M. cerebralis during our 2001–2003 survey of wild trout. For example, few T. tubifex worms were found at the McGinnis Run site where we observed the highest prevalence of infection. The McGinnis Run fish sample contained infected brook trout, rainbow trout, brown trout, and tiger trout. No T. tubifex worms were found at the Falling Springs Branch survey site where infected rainbow trout were collected. At Piney Creek

Of the 10 streams monitored during 2003, Galbraith Gap and Wildcat Run exhibited the lowest mean daily temperatures during the winter season (0–48C). In contrast, the lower-gradient, spring-fed streams remained consistently warmer (.48C) during the winter months (e.g., Falling Springs Branch; Figure 3). Mean daily temperatures never exceeded 208C in streams monitored during 2003. Big Fishing Creek and Spring Creek, two sites where M. cerebralis was detected, exhibited the coldest and warmest mean daily temperatures during the 2003 period of record. Mean daily temperature exceeded 108C earlier (March) at Falling Springs Branch than at most other sites. Temperatures at most streams exceeded a mean daily temperature of 108C between March 1 and May 1 during 2003. Big Spring Creek and Trindle Spring Run exceeded a mean daily temperature of 108C during most days of the year. M. cerebralis was detected in streams that exhibited both fluctuating (e.g., Big Fishing Creek) and stable (e.g., Falling Springs Branch) temperature regimens. The pH observed at our study sites ranged from 6.0 to 8.6. Higher pH, ANC, and conductivity values were observed among the lower-gradient, spring-fed streams (Table 4). M. cerebralis was detected at sites exhibiting pH values ranging from 6.9 to 8.6, and at conductivity values ranging from 83 to 607 lS/cm. Discussion The establishment and proliferation of M. cerebralis in a stream system is likely to be favored by a high abundance of susceptible trout, such as rainbow trout. In Pennsylvania, M. cerebralis appears to have persisted for decades among the highly abundant rainbow trout population of Falling Springs Branch. Population data, however, do not indicate that this population has been negatively affected by whirling disease. On the other hand, our detection of the parasite in Cedar Run (Clinton County) suggests that M. cerebralis has also persisted among a low-density

‹ e

If a fish culture facility discharged effluent into the stream within 11 km of the fish sampling site it is noted here by Y. We define Big Fishing Creek (upper) as reaches above the Tylersville fish culture facility and Big Fishing Creek (mid) as sites between the Tylersville and Lamar fish culture facilities. g When all pools test positive, apparent prevalence and confidence intervals cannot be calculated as described above. h The fish culture facility was not in operation at the time of fish sampling. f

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TABLE 2.—Trout population data (fish/ha) obtained during our 2001–2003 Pennsylvania survey and from the Pennsylvania Fish and Boat Commission for sites screened for M. cerebralis. All fish population data were collected within 3.0 km and 2 years of a parasite screening event. Stream a

Big Fishing Creek (upper) Big Fishing Creek (mid) Cherry Run Elk Creek (lower) Elk Creek (upper) Buffalo Runb Falling Springs Branchc Falling Springs Branch Falling Springs Branch Falling Springs Branch Cedar Run (Clinton County) Piney Creek McGinnis Run Little Fishing Creek Wallace Run (upper) Wallace Run (lower) Porcupine Run Porcupine Run Spring Creek (upper) Spring Creek (lower) Cedar Run (Centre County) Galbraith Gap Trindle Spring Run Trindle Spring Run Wildcat Run Fowlers Run Powdermill Run Furnace Run Laughlintown Run Tubmill Run a

b c

Year of survey

Brook trout

Rainbow trout

Brown trout

Total trout

Parasite detected

1978 2001 1979 1976 1979 1987 1984 1995 1997 2001 2002 2002 2001 1978 1979 1979 1976 1997 2000 2000 2002 2002 1997 2001 2001 2003 2001 2001 2001 2001

1,320 345 271 196 3,162 0 11 0 241 0 0 0 112 2,746 1,112 220 345 90 0 0 0 1,521 0 0 967 473 1,088 1,605 54 1,069

5 0 0 22 0 0 2,323 2,868 3,041 500 0 0 0 0 0 0 726 2,016 0 0 0 0 1,047 73 213 0 1,000 556 243 638

579 636 111 479 227 819 159 334 331 91 188 1,308 79 0 96 496 0 60 1,134 2,633 372 374 0 27 0 0 412 0 189 86

1,904 982 383 697 3,388 819 2,492 3,202 3,614 591 188 1,308 191 2,746 1,209 716 1,071 2,166 1,134 2,633 372 1,895 1,047 100 1,180 473 2,500 2,160 486 1,793

Yes No Yes Yes No Yes Yes Yes No Yes Yes Yes Yes No No No No No No No No No No No No No No No No No

This entry represents the average of three population estimates made approximately between 2.9 and 0.5 km above the Tylersville fish culture station. The first fish population survey conducted on Buffalo Run following the 1977 screening event was in 1987. The 1984, 1995, and 1997 estimates provided for Falling Springs Branch represent the average of three independent reach surveys within 2.0 km of our study site during each year listed.

population of brown trout, a species that exhibits resistance to infection and produces fewer myxospores to perpetuate the parasite life cycle than do infected rainbow trout (O’Grodnick 1979; Hedrick et al. 1999; Baldwin et al. 2000). Such disparate findings do not suggest a clear association between trout population composition and abundance and the occurrence of M. cerebralis among wild trout in Pennsylvania. Increased risk of M. cerebralis infection has been associated with lower stream gradients and lower elevation in the western United States (Schisler and Bergersen 2002). This association is likely attributable to the indirect effect of increased availability and stability of fine sediment habitat on the abundance of T. tubifex in this region. Most M. cerebralis detections in Pennsylvania occurred at low-gradient sites notably associated with fish culture facilities, riparian pastures, or both. Such point sources of organic enrichment have been associated with large colonies of T. tubifex, infection of wild trout in the eastern United States (Hulbert 1996) and elsewhere in the United States

(Yoder 1972; Horsch 1987; Modin 1998; Zendt and Bergersen 2000; Allen and Bergersen 2002), or both. In Pennsylvania, point sources of organic enrichment are more likely to be encountered along lower-gradient stream reaches near human developments than along forested mountain streams. In the exceptional case of McGinnis Run, a high-gradient stream with minimal fine sediment habitat, we observed a moderate prevalence (35%) of M. cerebralis. This site was located, however, a few hundred meters downstream of a fish culture facility. An important question is whether the parasitism of trout residing near such point sources in a Pennsylvania stream could lead to clinical whirling disease. O’Grodnick (1979) demonstrated that brief (72-h) exposures of sentinel brook trout and rainbow trout fry in Cedar Run (Clinton County) during winter resulted in clinical whirling disease. Larger, and presumably more resistant (Ryce et al. 2004) rainbow trout and brook trout exposed in Cedar Run for a 2-month period also developed clinical whirling disease. Given that the


FIGURE 2.—Relative abundance of the 16S mitochondrial DNA lineages of T. tubifex worms collected at five sites in Pennsylvania. Site codes are as follows: CR (CLT) ¼ Cedar Run (Clinton County), PC ¼ Piney Creek, MR ¼ McGinnis Run, CR (CEN) ¼ Cedar Run (Centre County), and FSB ¼ Falling Springs Branch. Worms were obtained from a small rearing pond that drained into McGinnis Run, from a small tributary that drained into Falling Spring Branch, and from the same sediment deposits sampled during our oligochaete distribution survey from the remaining three sites. At each site n ¼ 60 T. tubifex, except at the FSB tributary (n ¼ 48).

manifestation of clinical whirling disease among sentinel fish is an indicator of whirling disease outbreak among wild trout in the Intermountain West (Thompson et al. 1999; Downing et al. 2002; Thompson et al. 2002), it seems reasonable to speculate that outbreaks may have occurred among wild trout populations in Pennsylvania. The information gathered here regarding parasite prevalence and wild trout abundance is, however, limited by infrequent sampling and low detection levels and is insufficient to conclude whether clinical whirling disease outbreaks have or have not occurred among wild trout. Reproducing populations of brook trout once inhabited low-gradient, spring-fed streams throughout Pennsylvania. Such populations are virtually nonexistent today; their disappearance was scarcely documented. The collapse of a renowned brook trout fishery at Big Spring Creek (Cumberland County) during the period following the discovery of M. cerebralis in Pennsylvania (1958–1977) was previously attributed to organic pollution from two fish culture facilities (Kaeser 2004). Brook trout can develop whirling disease in a natural setting (Thompson et al. 1999; Vincent 2002), and infections can yield large numbers of myxospores (O’Grodnick 1979; Nehring and Walker 1996) supporting parasite population expansion. An unusually high prevalence of infection (77%) among the wild trout of Big Spring Creek was observed in 1978. Although it is impossible to prove or disprove whether an epizootic of whirling disease

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FIGURE 3.—Mean daily temperature trends for upper Spring Creek, Falling Springs Branch, and upper Big Fishing Creek during 2003. The three sites are located in Centre County, Franklin County, and Clinton County, Pennsylvania, respectively.

occurred in this system, several prominent risk factors historically converged at Big Spring Creek: a highly abundant population of susceptible brook trout and multiple point sources of gross organic enrichment overlapped in the headwaters of this low-gradient, sediment-rich stream. Moreover, hatchery-reared trout from the Big Spring Fish Culture Facility have never tested positive for M. cerebralis during routine inspections for this parasite (years 1974–1998), suggesting that the wild trout infected with M. cerebralis in 1977 and 1978 were indeed infected in Big Spring Creek proper. During the 2001–2003 survey, we found few populations of brook or rainbow trout that overlapped with populations of T. tubifex and suggest this is an important factor contributing to the limited parasite occurrence and lack of population-level effects currently attributable to M. cerebralis in Pennsylvania. The lack of overlap is due to the rarity of wild brook trout and rainbow trout populations in low-gradient streams, and the apparent restriction of T. tubifex to sites near organic point sources (Kaeser and Sharpe 2006). Research into the genetics of T. tubifex populations has revealed six unique clades or lineages in Europe and North America based on 16S mitochondrial DNA sequences (Sturmbauer et al. 1999; Beauchamp et al. 2001). These lineages frequently co-occur at stream locations, suggesting that the species morphologically distinguished as T. tubifex may actually be a cryptic, reproductively isolated species complex (Sturmbauer et al. 1999; Beauchamp et al. 2001; Baldo and Ferraguti 2005). Laboratory studies have shown that T. tubifex from lineages I and III are highly susceptible to

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TABLE 3.—Water quality variables measured at each study site during the 2001–2003 survey of Pennsylvania streams. Stream

pH

ANCa (leq/L)

Conductivity (lS/cm)

Wildcat Run Fowlers Run McGinnis Run Powdermill Run Furnace Run Laughlintown Run Tubmill Run Galbraith Gap Run Cedar Run (Centre County) Logan Branch Spring Creek (upper) Spring Creek (lower) Big Fishing Creek (upper) Big Fishing Creek (lower) Cedar Run (Clinton County) Duck Run Piney Creek Trindle Spring Run Big Spring Creek Falling Springs Branch

7.2 6.0 6.9 7.3 7.1 7.4 7.3 7.4 7.7 7.9 8.6 8.3 7.6 8.0 7.8 7.6 8.1 7.6 7.4 7.5

271 40 237 368 280 1,298 169 189 5,503 4,135 4,098 4,478 1,931 2,126 4,446 2,487 3,319 4,713 3,439 2,618

86 82 83 106 144 231 102 34 518 524 575 559 288 236 441 334 460 709 445 607

a

Acid neutralizing capacity.

infection by M. cerebralis, and T. tubifex from other lineages appear to be entirely resistant (e.g., lineages V and VI; Beauchamp et al. 2002, 2005). T. tubifex assemblages predominated by lineage I and III worms were found at several sites in Colorado where trout populations had been highly affected by whirling disease, suggesting that a predominance of lineages I and III may be a predisposing factor to disease outbreak (Beauchamp et al. 2002, 2005; Thompson et al. 2002; Nehring et al. 2003a). Lineage I and III worms were predominant at all five sites examined in Pennsylvania, suggesting that M. cerebralis establishment and propagation among these T. tubifex assemblages is not likely to be inhibited by host susceptibility. Dubey and Caldwell (2004) and Beauchamp et al. (2005) observed an association between the occurrence of lineage I and III worms and pool habitat and man-made structures. Of the five sites we examined, the McGinnis Run rearing pond was the only artificial impoundment sampled; the site was dominated by T. tubifex of lineage I. The other four sites were shallow depositional areas adjacent to streambanks. All five sites were located near riparian cattle pastures, fish culture facilities, or both, suggesting that lineage I and III T. tubifex may be associated with such sources of organic enrichment. Several researchers have reported an association between water temperature, the shedding of triactinomyxons from T. tubifex, and infection of trout in Intermountain West streams (El-Matbouli et al. 1999; Baldwin et al. 2000; Downing et al. 2002; MacConnell and Vincent 2002). In some cases, the optimal time for

triactinomyxon shedding appears to occur during late spring/summer and fall, when stream temperatures are within the range of 10–158C (Kerans and Zale 2002). High triactinomyxon concentrations have also been observed at much lower stream temperatures (Thompson and Nehring 2000; Nehring et al. 2003b). Considering the cooler temperature regimen observed at Big Fishing Creek and other forested, higher-gradient streams, we can only speculate that wild brook trout or brown trout, which typically emerge from the gravel between late February and April, might develop some physiological resistance (Ryce et al. 2004) prior to heavy exposure to the parasite if the peak release of these spores does in fact occur in the range of 10–158C. Given these assumptions, a lack of temporal overlap of susceptible juvenile trout and triactinomyxons seems an unlikely explanation for M. cerebralis epizootiology at Falling Springs Branch. Rainbow trout spawn in the spring in this stream and trout fry are likely to emerge between April and June, when mean daily temperatures are within the 10–158C range. More research is necessary before drawing any conclusions about disease dynamics related to the timing of juvenile trout emergence and release of triactinomyxons in Pennsylvania streams enzootic for M. cerebralis. Sandell et al. (2001) reported a correlation between high specific conductivity, T. tubifex abundance, and the prevalence of infection in sentinel trout. We identified a similar association between conductivity and total oligochaete abundance, and suggested that conductivity may serve as a gross indicator of T. tubifex abundance in some regions (Kaeser and Sharpe 2006). The presence of infected wild trout in McGinnis Run, however, is evidence that the transmission of M. cerebralis does occur in Pennsylvania streams that exhibit low conductivity. In summary, the available evidence does not readily suggest clear associations between the composition and abundance of resident trout populations, stream gradient, the genetic composition of T. tubifex populations, or conductivity and the pattern of occurrence of M. cerebralis among wild trout in Pennsylvania. Further investigation may, however, elucidate an association between these or other factors and M. cerebralis occurrence. We suggest the occurrence of M. cerebralis among wild trout in Pennsylvania may be best explained by the association between T. tubifex populations and anthropogenic point sources of organic enrichment such as fish culture facilities and riparian pastures that are more frequently encountered along low-gradient stream reaches. Susceptible T. tubifex worms must be present for the establishment of M. cerebralis among wild trout; we found susceptible lineages I and III T. tubifex downstream of sources of


organic enrichment. Regardless of differences that may exist in the temporal overlap of triactinomyxon spores and juvenile trout stages, if T. tubifex are not present to transmit the parasite to wild trout, then the infection cycle will be broken. If widespread spatial overlap between T. tubifex worms and highly susceptible species such as brook trout or rainbow trout is lacking, it seems unlikely that M. cerebralis could proliferate throughout a stream system, leading to disease outbreak and detectable trout population decline. Further investigation of the spatial distribution and abundance of T. tubifex in Pennsylvania streams that are enzootic for M. cerebralis and contain wild brook trout or rainbow trout populations should be conducted. Acknowledgments The authors thank John Coll and the staff of the U. S. Fish and Wildlife Service Fish Health Laboratory in Lamar, Pennsylvania, for their assistance with the examination of fish samples, and Ana Baya of the Animal Health Laboratory, Maryland Department of Agriculture, for microscopic examination of cranial histologic sections. We also thank the many landowners who granted access to property to conduct field work, Joe Merritt and the Powdermill Nature Reserve for providing housing for our field crew, and K. Stark and B. Niewinski of the Pennsylvania Fish and Boat Commission for their assistance. References Allen, M. B., and E. P. Bergersen. 2002. Factors influencing the distribution of Myxobolus cerebralis, the causative agent of whirling disease, in the Cache la Poudre River, Colorado. Diseases of Aquatic Organisms 49:51–60. Andree, K. B., E. MacConnell, and R. P. Hedrick. 1998. A polymerase chain reaction test for detection of Myxobolus cerebralis, the causative agent of salmonid whirling disease in fish, and a comparison to existing detection techniques. Diseases of Aquatic Organisms 34:145–154. Baldo, L., and M. Ferraguti. 2005. Mixed reproductive strategy in Tubifex tubifex (Oligochaeta, Tubificidae)? Journal of Experimental Zoology 303A:168–177. Baldwin, T. J., E. R. Vincent, R. M. Silflow, and D. Stanek. 2000. Myxobolus cerebralis infection in rainbow trout (Oncorhynchus mykiss) and brown trout (Salmo trutta) exposed under natural stream conditions. Journal of Veterinary Diagnostic Investigation 12:312–321. Bartholomew, J. L., and P. W. Reno. 2002. Review: the history and dissemination of whirling disease. Pages 3– 24 in J. L. Bartholomew and J. C. Wilson editors. Whirling disease: reviews and current topics. American Fisheries Society, Symposium 29, Bethesda, Maryland. Beauchamp, K. A., R. D. Kathman, T. S. McDowell, and R. P. Hedrick. 2001. Molecular phylogeny of tubificid oligochaetes with special emphasis on Tubifex tubifex

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and anadromous salmonids. Journal of Aquatic Animal Health 13:142–150. Schisler, G. J., and E. P. Bergersen. 2002. Evaluation of risk of high-elevation Colorado waters to the establishment of Myxobolus cerebralis. Pages 33–41 in J. L. Bartholomew and J. C. Wilson editors. Whirling disease: reviews and current topics. American Fisheries Society, Symposium 29, Bethesda, Maryland. Sturmbauer, C., G. B. Opadiya, H. Niederstatter, A. Riedmann, and R. Dallinger. 1999. Mitochondrial DNA reveals cryptic oligochaete species differing in cadmium resistance. Molecular Biology and Evolution 16:967–974. Thoesen, J. C. 2004. AFSFHS (American Fisheries SocietyFish Health Section). FHS blue book: suggested procedures for the detection and identification of certain finfish and shellfish pathogens, 2004 edition. AFSFHS, Bethesda, Maryland. Thompson, K. G., R. B. Nehring, D. C. Bowden, and T. Wygant. 1999. Field exposure of seven species or subspecies of salmonids to Myxobolus cerebralis in the Colorado River, Middle Park, Colorado. Journal of Aquatic Animal Health 11:312–329. Thompson, K. G., and R. B. Nehring. 2000. A simple technique used to filter and quantify the actinospore of Myxobolus cerebralis and determine its seasonal abundance in the Colorado River. Journal of Aquatic Animal Health 12:316–323. Thompson, K. G., R. B. Nehring, D. C. Bowden, and T. Wygant. 2002. Response of rainbow trout Oncorhynchus mykiss to exposure to Myxobolus cerebralis above and below a point source of infectivity in the upper Colorado River. Diseases of Aquatic Organisms 49:171–178. Van Deventer, J. S., and W. S. Platts. 1985. A computer software program system for entering, managing, and analyzing fish capture data from streams. U.S. Forest Service, Ogden, Utah. Vincent, E. R. 1996. Whirling disease and wild trout: the Montana experience. Fisheries 21(6):32–33. Vincent, E. R. 2002. Relative susceptibility of various salmonids to whirling disease with emphasis on rainbow and cutthroat trout. Pages 109–115 in J. L. Bartholomew and J. C. Wilson editors. Whirling disease: reviews and current topics. American Fisheries Society, Symposium 29, Bethesda, Maryland. Wagner, E. J. 2002. Whirling disease prevention, control, and management: a review. Pages 217–225 in J. L. Bartholomew and J. C. Wilson editors. Whirling disease: reviews and current topics. American Fisheries Society, Symposium 29, Bethesda, Maryland. Williams, C. J., and C. M. Moffitt. 2001. A critique of methods of sampling and reporting pathogens in populations of fish. Journal of Aquatic Animal Health 13:300–309. Yoder, W. G. 1972. The spread of Myxosoma cerebralis into native trout populations in Michigan. Progressive FishCulturist 34:103–106. Zendt, J. S., and E. P. Bergersen. 2000. Distribution and abundance of the aquatic oligochaete host Tubifex tubifex for the salmonid whirling disease parasite Myxobolus cerebralis in the upper Colorado River basin. North American Journal of Fisheries Management 20:502–512.