Christine M. Moffitt

A Critique of Methods of Sampling and Reporting Pathogens in Populations of Fish

Abstract We used maximum likelihood methods to estimate an observed or apparent prevalence for a pathogen in a pooled sample of fish and here provide the program code for such calculations using commonly available statistical software. To illustrate the characteristics and variability of prevalence estimates from pooled samples, we explored the relationships among pathogen prevalence, sample size, and method of pooling samples. We calculated the average width of confidence intervals and the mean square error of the prevalence estimator for samples from populations with pathogen prevalence ranging from 1% to 90% using several pooling strategies for samples of 30 and 60 fish. As an illustration, we calculated the confidence interval and apparent prevalence of Myxobolus cerebralis in samples of fish from Utah screened with pooled sampling strategies. When all pools were positive, the apparent prevalence was 100%, but the bounds of the confidence interval ranged from 8% to 100%. Interpretations of data sets t...

Variation in Infections of Myxobolus cerebralis in Field-Exposed Cutthroat and Rainbow Trout in Idaho

Abstract We evaluated the severity and extent of infection by Myxobolus cerebralis in replicated groups of same-age Yellowstone cutthroat trout Oncorhynchus clarki bouvieri and Kamloops rainbow trout O. mykiss exposed to natural river conditions at four sites along a 25-km reach of the South Fork of the Boise River. Groups of fry were each exposed for 10 d in June, July, or August and then removed from the river and reared in specific-pathogen-free water under controlled laboratory conditions for 84 d. At the end of the study, surviving fish were examined for clinical signs of disease, and fish heads were evaluated by histology for the presence of characteristic pathology and then scored for the extent of pathology on a five-point scale. The mean histological scores and percent of fish testing positive in both species were similar within each site but varied across the study sites from no infection in fish at the uppermost site to 90–100% testing positive at the lowermost site. Cutthroat trout with modera...

Survival and Passage of Ingested New Zealand Mudsnails through the Intestinal Tract of Rainbow Trout

Abstract We conducted laboratory trials to determine the transit time and survival of New Zealand mudsnails Potamopyrgus antipodarum in the gastrointestinal tract of rainbow trout Oncorhynchus mykiss. To assess the rate of snail passage, we force-fed groups of fish a known quantity of snails and then held them in tanks. At selected intervals we removed individual fish from the test tanks and recorded the number of snails, their condition (live or dead), and their location in the gastrointestinal tract (stomach, anterior intestine, and posterior intestine). Feces were removed from tanks and examined for live snails. We repeated evaluations of passage rate and snail survival to determine the effects of varying the number of snails ingested, fish size, snail size, and feeding a commercial diet to fish after snail ingestion. We plotted and modeled gut evacuation using a stochastic model for ordinal data to consider each test variable. Snail passage rates were faster in fish that were fed smaller snails. Surpr...

A Quantitative Enzyme-Linked Immunosorbent Assay and Filtration-Based Fluorescent Antibody Test as Potential Tools to Screen Broodstock for Infection with Flavobacterium psychrophilum

There is strong evidence that Flavobacterium psychrophilum, the etiologic agent of coldwater disease, is transmitted vertically; it has been hypothesized that disease management at hatchery facilities can be improved through broodstock screening and implementation of culling programs. This paper describes the development of two assays used to screen broodstock tissues (kidney and ovarian fluid) for the presence of F. psychrophilum. Four monoclonal antibodies (MAbs) were generated against outer membrane preparations of F. psychrophilum strain CSF (Clear Springs Foods) 259-93. Of these, MAb FL43 was selected for assay development; this MAb reacted with 67 isolates of F. psychrophilum but exhibited no reaction with two strains of F. columnare or single strains of F. pectinovorum, F. aquatile, F. branchiophilum, and F. saccharophilum. An enzyme-linked immunosorbent assay (ELISA) was developed using MAb FL43 as the capture antibody and MAb FL43 conjugated to horseradish peroxidase (enzyme number 1.11.1.7; IUBMB 1992) as the secondary detection antibody. The ELISA had a lower F. psychrophilum detection boundary of approximately 1.6 X 10(3) colony-forming units (CFU)/mL in kidney tissue homogenates spiked with known bacterial concentrations. Asymptomatic broodstock of coho salmon Oncorhynchus kisutch (n = 50 fish) were sampled, and 100% tested positive for infection by ELISA analysis of kidney tissue; bacterial load was estimated at 2.0 x 10(3) to 9.4 x 10(3) CFU/mL. Ovarian fluid was also collected from these same coho salmon as well as from broodstock of rainbow trout O. mykiss; however, the ELISA proved to be unsuitable for use with ovarian fluid. A filtration-based fluorescent antibody test (FAT) was subsequently developed by conjugating MAb FL43 to Alexa Fluor 488. This FAT was able to detect F. psychrophilum in 74% of ovarian fluid samples collected from coho salmon and 42% of ovarian fluid samples from rainbow trout. Interestingly, yellow-pigmented bacteria were isolated on culture plates from 100% of kidney and ovarian fluid samples. All yellow-pigmented colonies were tested by polymerase chain reaction, and 100% of the coho salmon and rainbow trout were confirmed positive for infection with F. psychrophilum.

Profile of Microflora of the Posterior Intestine of Chinook Salmon before, during, and after Administration of Rations with and without Erythromycin

Abstract We describe the resident heterotrophic aerobic microflora of the salmonid posterior intestine before, during, and after the administration of rations with erythromycin in a hatchery raceway environment. We compare the profiles of medicated Chinook salmon Oncorhynchus tshawytscha with those of control fish that were not fed erythromycin. The combined counts of bacteria and yeasts per gram of fish intestine originating from four upstream raceways ranged from 3.0 × 102 to 9.6 × 105 colony-forming units (CFU) over the study period. Yeasts were commonly identified in the gut, and abundances ranged from 0% to more than 80% of the CFU. Erythromycin therapy decreased the total microbial population and altered the bacterial diversity in the gut during treatment. The intestinal microbial populations in fish medicated with erythromycin increased rapidly after treatment ceased, and by 25 d after treatment the CFU were similar in samples from both medicated and control fish populations. Of 325 isolates from f...

Palatability of Feed Containing Different Concentrations of Erythromycin Thiocyanate to Chinook Salmon

Abstract The feeding behavior of juvenile chinook salmon (Oncorhynchus tshawytscha) was observed to determine the palatability of feed containing the antibiotic erythromycin thiocyanate. Individual fish in aquaria were offered feed pellets containing 0, 6.7, 10.0, or 12.0 μg erythromycin/mg feed. Compared with fish offered a diet without erythromycin, fish offered pellets with antibiotic took a significantly longer time to strike each pellet, ejected individual pellets more often, took a longer time to consume each pellet, and consumed fewer total pellets. The high rejection rate of the diets containing 10.0 and 12.0 μg erythromycin/mg was attributed to the unpalatability of the drug at these concentrations. Fish took more time to strike the drug-treated versus the antibiotic-free (control) pellets even during the first feeding trial, indicating that both smell and taste may play roles in determining feed consumption behavior. The effectiveness of feeding erythromycin thiocyanate to juvenile chinook salmo...

Models of stream habitat characteristics associated with tubificid populations in an intermountain watershed

Habitat assessments were conducted in an intermountain watershed at three spatial extents to explore ways to predict the presence of tubificid oligochaetes likely to support the parasite Myxobolus cerebralis, cause of salmonid whirling disease. Stream reaches with six different reach slope characteristics were selected using GIS. The aquatic habitat in 60 reaches selected at random was measured and classified into distinct habitat units. Within the habitat units, areas of microhabitat with depositional fine sediments were chosen, measured, and core samples were removed to characterize the sediments and benthic oligochaetes. Two tubificids, Tubifex spp. and Limnodrilus hoffmeisteri, were abundant and co-occurred in silt-clay and fine sand sediments in these habitats. Models were posed and tested to predict the presence and relative abundance of tubificids using habitat characteristics from the three spatial extents: reach, habitat unit, and microhabitat. At the reach extent, tubificids were associated with low-reach slope and with slow water habitats. Within habitat units, tubificids were associated with higher percentages of fine sediments and higher stream width:depth ratios. In microhabitat cores, the presence of silt-clay sediments was positively associated with higher average stream width:depth ratios. Since ecological relationships are often scale dependent and stream systems have a natural hierarchy, predictive habitat models such as these that use measures from several scales may help researchers and managers more efficiently identify and quantify aquatic communities at highest risk of infection by the M. cerebralis parasite.

Estimation of fish and wildlife disease prevalence from imperfect diagnostic tests on pooled samples with varying pool sizes

Abstract Methods of estimating disease or parasite prevalence in free-ranging and some captive fish and wildlife populations are frequently lacking in precision due to limited numbers of observations and different assay procedures. Recently statistical methods and software programs have been developed to use Bayesian and other methods to obtain estimates of disease prevalence from diagnostic tests in which sensitivity and/or specificity is not perfect (imperfect) and with sampling schemes using pooled samples. However, these published methods and software programs that consider pooled data sampling have generally considered the case of one uniform pool size for all samples. We present a method for estimating disease prevalence from imperfect diagnostic tests with pooled data collected from a variety of pool sizes. We use a Bayesian approach and obtain a sample from the posterior distribution of prevalence, sensitivity, and specificity, using an MCMC sampling algorithm implemented in the WINBUGS statistical package. We illustrate the use of these methods with three examples and perform efficiency calculations to investigate the performance of these estimators relative to maximum likelihood estimators that assume perfect diagnostic tests. Our results illustrate that the estimates produced from these methods adjust for imperfect tests, and are often more efficient than estimates assuming perfect tests, except in some situations when there is not much prior information on diagnostic test sensitivity and specificity.

Accumulation and Depletion of Orally Administered Erythromycin Thiocyanate in Tissues of Chinook Salmon

Abstract Juvenile chinook salmon Oncorhynchus tshawytscha were fed erythromycin thiocyanate for 21 d at a daily therapeutic dosage of either 0.05 or 0.1 g erythromycin/kg body weight. We analyzed samples of liver, spleen, kidney, red muscle, white muscle, and plasma removed from fish at intervals during and after drug administration to determine the concentration of erythromycin present. On all but one sampling date, the kidney had the highest mean concentration (weight of drug per unit of tissue weight) among the tissues sampled. The mean concentrations in all tissues and plasma offish fed at the daily dosage of 0.05 g/kg did not differ significantly over time while the drug was fed. However, erythromycin accumulated over time in the liver, spleen, kidney, red muscle, and white muscle offish fed at 0.1 g/kg; concentrations of the drug peaked on the last day of antibiotic administration. Doubling the dosage from 0.05 to 0.1 g/kg resulted in a 4–10-fold increase in erythromycin concentration in tissues, an...

Proximate Composition and Energy Density of Stream-Maturing Adult Steelhead during Upstream Migration, Sexual Maturity, and Kelt Emigration

AbstractIn anadromous salmonids, muscle tissue provides the primary source of energy to support spawning migrations and spawning. We examined changes in the lipid content, protein content, and energy density of white muscle collected from stream-maturing Snake River–Columbia River steelhead Oncorhynchus mykiss during several phases of reproduction. At a basinwide scale, we estimated that between early freshwater entry and postspawning (kelt) emigration, the lipid content of white muscle was reduced by 94% to levels less than 1% of wet tissue weight. Lipid was depleted more rapidly than protein during the reproductive cycle and afterward provided the only remaining somatic energy source for the postspawning migration. We found that protein content was consistently higher in sexually mature male steelhead than in females, suggesting that energy allocation prior to reproduction varies between the sexes. In kelts, the lipid content, protein content, and energy density of white muscle were significantly higher...