Dorothy M. Chase

Comparison of the Membrane-Filtration Fluorescent Antibody Test, the Enzyme-Linked Immunosorbent Assay, and the Polymerase Chain Reaction to Detect Renibacterium Salmoninarum in Salmonid Ovarian Fluid

Ovarian fluid samples from naturally infected chinook salmon (Oncorhynchus tshawytscha) were examined for the presence of Renibacterium salmoninarum by the membrane-filtration fluorescent antibody test (MF-FAT), an antigen capture enzyme-linked immunosorbent assay (ELISA), and a nested polymerase chain reaction (PCR). On the basis of the MF-FAT, 64% (66/103) samples contained detectable levels of R. salmoninarum cells. Among the positive fish, the R. salmoninarum concentrations ranged from 25 cells/ml to 4.3 × 109 cells/ml. A soluble antigenic fraction of R. salmoninarum was detected in 39% of the fish (40/103) by the ELISA. The ELISA is considered one of the most sensitive detection methods for bacterial kidney disease in tissues, yet it did not detect R. salmoninarum antigen consistently at bacterial cell concentrations below about 1.3 × 104 cells/ml according to the MF-FAT counts. When total DNA was extracted and tested in a nested PCR designed to amplify a 320-base-pair region of the gene encoding a soluble 57-kD protein of R. salmoninarum, 100% of the 100 samples tested were positive. The results provided strong evidence that R. salmoninarum may be present in ovarian fluids thought to be free of the bacterium on the basis of standard diagnostic methods.

Detection and quantification of Renibacterium salmoninarum DNA in salmonid tissues by real-time quantitative polymerase chain reaction analysis.

Renibacterium salmoninarum is an important salmonid pathogen that is difficult to culture. We developed and assessed a real-time, quantitative, polymerase chain reaction (qPCR) assay for the detection and enumeration of R. salmoninarum. The qPCR is based on TaqMan technology and amplifies a 69-base pair (bp) region of the gene encoding the major soluble antigen (MSA) of R. salmoninarum. The qPCR assay consistently detected as few as 5 R. salmoninarum cells per reaction in kidney tissue. The specificity of the qPCR was confirmed by testing the DNA extracts from a panel of microorganisms that were either common fish pathogens or reported to cause false-positive reactions in the enzyme-linked immunosorbent assay (ELISA). Kidney samples from 38 juvenile Chinook salmon (Oncorhynchus tshawytscha) in a naturally infected population were examined by real-time qPCR, a nested PCR, and ELISA, and prevalences of R. salmoninarum detected were 71, 66, and 71%, respectively. The qPCR should be a valuable tool for evaluating the R. salmoninarum infection status of salmonids.

Comparison of Traditional and Molecular Methods for Detection of Renibacterium Salmoninarum

Bacterial kidney disease (BKD) caused by Renibacterium salmoninarum occurs in most parts of the world where wild or cultured salmonid fishes are present. Several extensive reviews have been written on the pathogen and the disease (Fryer and Sanders, 1981; Austin and Austin, 1987; Elliott et al., 1989; Evelyn, 1993; Evenden et al., 1993; Fryer and Lannan, 1993). Bacterial kidney disease can cause serious mortality in juvenile salmonids in both fresh water and seawater, and also in prespawning adults. Although the chronic nature of the disease has hindered accurate estimates of fish losses, particularly in feral fish populations, BKD is one the most important bacterial diseases affecting cultured salmonids, with reported losses as high as 80% in stocks of Pacific salmon (Oncorhynchus spp.) and 40% in stocks of Atlantic salmon (Salmo salar) (Evenden et al., 1993).

Distribution of Pacific lamprey Entosphenus tridentatus in watersheds of Puget Sound Based on smolt monitoring data

Abstract Lamprey populations are in decline worldwide and the status of Pacific lamprey (Entosphenus tridentatus) is a topic of current interest. They and other lamprey species cycle nutrients and serve as prey in riverine ecosystems. To determine the current distribution of Pacific lamprey in major watersheds flowing into Puget Sound, Washington, we sampled lamprey captured during salmonid smolt monitoring that occurred from late winter to mid-summer. We found Pacific lamprey in 12 of 18 watersheds and they were most common in southern Puget Sound watersheds and in watersheds draining western Puget Sound (Hood Canal). Two additional species, western brook lamprey (Lampetra richardsoni) and river lamprey (L. ayresii) were more common in eastern Puget Sound watersheds. Few Pacific lamprey macrophthalmia were found, suggesting that the majority of juveniles migrated seaward during other time periods. In addition, “dwarf” adult Pacific lamprey (< 300 mm) were observed in several watersheds and may represent an alternate life history for some Puget Sound populations. Based on genetic data, the use of visual techniques to identify lamprey ammocoetes as Entosphenus or Lampetra was successful for 97% (34 of 35) of the samples we evaluated.

Testing of candidate non-lethal sampling methods for detection of Renibacterium salmoninarum in juvenile Chinook salmon Oncorhynchus tshawytscha

Non-lethal pathogen testing can be a useful tool for fish disease research and management. Our research objectives were to determine if (1) fin clips, gill snips, surface mucus scrapings, blood draws, or kidney biopsies could be obtained non-lethally from 3 to 15 g Chinook salmon Oncorhynchus tshawytscha, (2) non-lethal samples could accurately discriminate between fish exposed to the bacterial kidney disease agent Renibacterium salmoninarum and non-exposed fish, and (3) non-lethal samples could serve as proxies for lethal kidney samples to assess infection intensity. Blood draws and kidney biopsies caused ≥5% post-sampling mortality (Objective 1) and may be appropriate only for larger fish, but the other sample types were non-lethal. Sampling was performed over 21 wk following R. salmoninarum immersion challenge of fish from 2 stocks (Objectives 2 and 3), and nested PCR (nPCR) and real-time quantitative PCR (qPCR) results from candidate non-lethal samples were compared with kidney tissue analysis by nPCR, qPCR, bacteriological culture, enzyme-linked immunosorbent assay (ELISA), fluorescent antibody test (FAT) and histopathology/immunohistochemistry. R. salmoninarum was detected by PCR in >50% of fin, gill, and mucus samples from challenged fish. Mucus qPCR was the only non-lethal assay exhibiting both diagnostic sensitivity and specificity estimates>90% for distinguishing between R. salmoninarum-exposed and non-exposed fish and was the best candidate for use as an alternative to lethal kidney sample testing. Mucus qPCR R. salmoninarum quantity estimates reflected changes in kidney bacterial load estimates, as evidenced by significant positive correlations with kidney R. salmoninarum infection intensity scores at all sample times and in both fish stocks, and were not significantly impacted by environmental R. salmoninarum concentrations.

Temporal Genetic Monitoring of Hybridization between Native Westslope Cutthroat Trout and Introduced Rainbow Trout in the Stehekin River, Washington

Abstract Introgressive hybridization with introduced rainbow trout (RBT) (Oncorhynchus mykiss) has led to the loss of native cutthroat trout species (O. clarkii) throughout their range, creating conservation concerns. Monitoring temporal hybridization trends provides resource managers with a tool for determining population status and information for establishing conservation goals for native cutthroat trout. In this study, we re-sampled six locations in 2010 within the Stehekin River watershed, North Cascades National Park, which were originally sampled between 1999 and 2003. We used genetic markers to monitor changes in hybridization levels between sampling periods in the native westslope cutthroat trout (WCT) (O. c. lewisi) stemming from past RBT introductions. Additionally, two new locations from the lower Stehekin drainage were added to the baseline data. We found that the frequency of WCT, RBT, and their hybrids was not significantly different between monitoring periods, but that RBT allele frequencies decreased in two locations and increased in one location. We also found a consistent, substantial reduction in the frequency of RBT alleles over the monitoring period in the Stehekin River upstream of Bridge Creek (SR3) compared to the Stehekin River downstream of Bridge Creek (SR1 -2) and within lower Bridge Creek (BR1) although these three locations are confined to a small geographic area (approximately 5 km). Ecological and/or evolutionary processes likely restrict the dispersal of RBT alleles in the Stehekin River upstream of Bridge Creek.

Hybridization between Yellowstone Cutthroat Trout and Rainbow Trout Alters the Expression of Muscle Growth-Related Genes and Their Relationships with Growth Patterns.

Hybridization creates novel gene combinations that may generate important evolutionary novelty, but may also reduce existing adaptation by interrupting inherent biological processes, such as genotype-environment interactions. Hybridization often causes substantial change in patterns of gene expression, which, in turn, may cause phenotypic change. Rainbow trout (Oncorhynchus mykiss) and cutthroat trout (O. clarkii) produce viable hybrids in the wild, and introgressive hybridization with introduced rainbow trout is a major conservation concern for native cutthroat trout. The two species differ in body shape, which is likely an evolutionary adaptation to their native environments, and their hybrids tend to show intermediate morphology. The characterization of gene expression patterns may provide insights on the genetic basis of hybrid and parental morphologies, as well as on the ecological performance of hybrids in the wild. Here, we evaluated the expression of eight growth-related genes (MSTN-1a, MSTN-1b, MyoD1a, MyoD1b, MRF-4, IGF-1, IGF-2, and CAST-L) and the relationship of these genes with growth traits (length, weight, and condition factor) in six line crosses: both parental species, both reciprocal F1 hybrids, and both first-generation backcrosses (F1 x rainbow trout and F1 x cutthroat trout). Four of these genes were differentially expressed among rainbow, cutthroat, and their hybrids. Transcript abundance was significantly correlated with growth traits across the parent species, but not across hybrids. Our findings suggest that rainbow and cutthroat trout exhibit differences in muscle growth regulation, that transcriptional networks may be modified by hybridization, and that hybridization disrupts intrinsic relationships between gene expression and growth patterns that may be functionally important for phenotypic adaptations.

Distribution and seasonal differences in Pacific Lamprey and Lampetra spp eDNA across 18 Puget Sound watersheds

Lampreys have a worldwide distribution, are functionally important to ecological communities and serve significant roles in many cultures. In Pacific coast drainages of North America, lamprey populations have suffered large declines. However, lamprey population status and trends within many areas of this region are unknown and such information is needed for advancing conservation goals. We developed two quantitative PCR-based, aquatic environmental DNA (eDNA) assays for detection of Pacific Lamprey (Entosphenus tridentatus) and Lampetra spp, using locked nucleic acids (LNAs) in the probe design. We used these assays to characterize the spatial distribution of lamprey in 18 watersheds of Puget Sound, Washington, by collecting water samples in spring and fall. Pacific Lamprey and Lampetra spp were each detected in 14 watersheds and co-occurred in 10 watersheds. Lamprey eDNA detection rates were much higher in spring compared to fall. Specifically, the Pacific Lamprey eDNA detection rate was 3.5 times higher in spring and the Lampetra spp eDNA detection rate was 1.5 times higher in spring even though larval lamprey are present in streams year-round. This significant finding highlights the importance of seasonality on eDNA detection. Higher stream discharge in the fall likely contributed to reduced eDNA detection rates, although seasonal life history events may have also contributed. These eDNA assays differentiate Pacific Lamprey and Lampetra spp across much of their range along the west coast of North America. Sequence analysis indicates the Pacific Lamprey assay also targets other Entosphenus spp and indicates the Lampetra spp assay may have limited or no capability of detecting Lampetra in some locations south of the Columbia River Basin. Nevertheless, these assays will serve as a valuable tool for resource managers and have direct application to lamprey conservation efforts, such as mapping species distributions, occupancy modeling, and monitoring translocations and reintroductions.