Genetic characterization of Flavobacterium columnare isolates from the Pacific Northwest, USA.

Flavobacterium columnare is the causative agent of columnaris disease. Previous work has demonstrated a high degree of genetic variability among F. columnare isolates, identifying 4 genetic groups (GGs) with some host associations. Herein, a total of 49 F. columnare isolates were characterized, the majority of which were collected from 15 different locations throughout the US Pacific Northwest. Most isolates were collected from 2015-2018 and originated from disease outbreaks in salmonid hatcheries and rearing ponds, sturgeon hatcheries and ornamental fish. Other isolates were part of collections recovered from 1980-2018. Initial identification was confirmed by F. columnare species-specific qPCR. Study isolates were further characterized using a multiplex PCR that differentiates between the 4 currently recognized F. columnare GGs. Multiplex PCR results were supported by repetitive sequence-mediated PCR fingerprinting and gyrB sequence analysis. F. columnare GG1 was the most prevalent (83.7%, n = 41/49), represented by isolates from salmonids (n = 32), white sturgeon (n = 2), channel catfish (n = 1), ornamental goldfish (n = 1), koi (n = 3), wild sunfish (n = 1) and 1 unknown host. Six isolates (12.2%, n = 6/49) were identified as GG3, which were cultured from rainbow trout (n = 3) and steelhead trout (n = 3). Two isolates were identified as GG2 (4.1%, n = 2/49) and were from ornamental fish. No GG4 isolates were cultured in this study. The biological significance of this genetic variability remains unclear, but this variation could have significant implications for fish health management. The results from this study provide baseline data for future work developing strategies to ameliorate columnaris-related losses in the US Pacific Northwest.

Relationship between temperature and Ceratomyxa shasta -induced mortality in Klamath River salmonids.

Water temperature influences almost every biological and physiological process of salmon, including disease resistance. In the Klamath River (California), current thermal conditions are considered sub-optimal for juvenile salmon. In addition to borderline temperatures, these fish must contend with the myxozoan parasite Ceratomyxa shasta , a significant cause of juvenile salmonid mortality in this system. This paper presents 2 studies, conducted from 2007 to 2010, that examine thermal effects on C. shasta -induced mortality in native Klamath River Chinook ( Oncorhynchus tshawytscha ) and coho ( Oncorhynchus kisutch ) salmon. In each study, fish were exposed to C. shasta in the Klamath River for 72 hr and then reared in the laboratory under temperature-controlled conditions. The first study analyzed data collected from a multi-year monitoring project to asses the influence of elevated temperatures on parasite-induced mortality during the spring/summer migration period. The second study compared disease progression in both species at 4 temperatures (13, 15, 18, and 21 C) representative of spring/summer migration conditions. Both studies demonstrated that elevated water temperatures consistently resulted in higher mortality and faster mean days to death. However, analysis of data from the multi-year monitoring showed that the magnitude of this effect varied among years and was more closely associated with parasite density than with temperature. Also, there was a difference in the timing of peak mortality between species; Chinook incurred high mortalities in 2008 and 2009, whereas coho was greatest in 2007 and 2008. As neither temperature nor parasite density can be easily manipulated, management strategies should focus on disrupting the overlap of this parasite and its obligate hosts to improve emigration success and survival of juvenile salmon in the Klamath River.

Density of the waterborne parasite Ceratomyxa shasta and its biological effects on salmon.

The myxozoan parasite Ceratomyxa shasta is a significant pathogen of juvenile salmonids in the Pacific Northwest of North America and is limiting recovery of Chinook (Oncorhynchus tshawytscha) and coho (O. kisutch) salmon populations in the Klamath River. We conducted a 5-year monitoring program that comprised concurrent sentinel fish exposures and water sampling across 212 river kilometers of the Klamath River. We used percent mortality and degree-days to death to measure disease severity in fish. We analyzed water samples using quantitative PCR and Sanger sequencing, to determine total parasite density and relative abundance of C. shasta genotypes, which differ in their pathogenicity to salmonids. We detected the parasite throughout the study zone, but parasite density and genetic composition fluctuated spatially and temporally. Chinook and coho mortality increased with density of their specific parasite genotype, but mortality-density thresholds and time to death differed. A lethality threshold of 40% mortality was reached with 10 spores liter(-1) for Chinook but only 5 spores liter(-1) for coho. Parasite density did not affect degree-days to death for Chinook but was negatively correlated for coho, and there was wider variation among coho individuals. These differences likely reflect the different life histories and genetic heterogeneity of the salmon populations. Direct quantification of the density of host-specific parasite genotypes in water samples offers a management tool for predicting host population-level impacts.

Surveillance for Ceratomyxa shasta in the Puget Sound watershed, Washington.

Discovery of fish exhibiting clinical signs of ceratomyxosis in Washington State prompted concern over the potential impact of the myxozoan parasite Ceratomyxa shasta on native stocks of steelhead Oncorhynchus mykiss (anadromous rainbow trout). To investigate these concerns, a survey of 16 freshwater systems within the Puget Sound watershed, including Lake Washington, was conducted by sentinel exposure of susceptible fish (cutthroat trout O. clarkii and rainbow trout). Fish were exposed for 7 d during September 2003 and May 2004 and then were returned to a holding facility for monitoring of disease signs. Mortality caused by the parasite occurred only in the exposure group held at the University of Washington Hatchery, which receives its water from Portage Bay of Lake Washington. Fish from all other sites were negative for C. shasta, both visually and by polymerase chain reaction (PCR) assay, except for a single fish held at the Tumwater Falls Hatchery in September 2003. A single deformed spore was detected in that fish, but infection could not be confirmed by PCR and the parasite was not detected from any other fish held at that site during either the September or the May exposure. From these results, we conclude that C. shasta is not likely to have contributed significantly to the decline of steelhead populations throughout Puget Sound.

A new myxozoan from feral goldfish (Carassius auratus).

In February 2004, a mass die-off of common goldfish Carassius auratus L., presumptively caused by bacterial coldwater disease (Flavobacterium psychrophilum), occurred at Fern Ridge Reservoir, Oregon. A range of size classes was affected, but all mature fish were female and all fish were infected with a single myxozoan, Chloromyxum auratum n. sp. No histological changes were observed associated with the parasite. Infection was represented by mictosporic plasmodia and free-floating spores in the gall bladder. Parasite spores were nearly spherical, 13.6 microm long x 12.6 microm wide x 13.1 microm thick, and possessed 4 equal-sized polar capsules. Spores had a coglike appearance in apical view because of distinct ridges 2.1 microm high protruding from the valve cells. There were 6-9 extrasutural ridges per valve (15-20 ridges per spore), aligned along the longitudinal axis, with some branching, and convergence at both poles. Morphologically, spores identified most closely with Chloromyxum cristatum Léger, 1906; however, 18S rDNA sequence data indicated only 97.5% similarity over 2,076 bp with Chloromyxum cyprini, the only synonym of C. cristatum for which DNA data are available; additional sequence data may reveal the other synonyms to be distinct species. This is the first record of a species of Chloromyxum from goldfish.

Survival of Lost River suckers (Deltistes luxatus) challenged with Flavobacterium columnare during exposure to sublethal ammonia concentrations at ph 9.5.

The Lost River sucker (Deltistes luxatus) is a federally listed, endangered species inhabiting the hypereutrophic waters of Upper Klamath Lake in southern Oregon, USA. High pH (> or =10) and elevated ammonia concentrations (> or =1 mg NH(3)-N/L) often occur during blooms of cyanobacteria (Aphanizomenon flos-aquae) in the lake, with major fish kills sometimes following a mid- or late-summer "crash" of the cyanobacterial population. Previous histopathology analyses and bacterial sampling indicated that infections of the pathogenic bacterium Flavobacterium columnare might contribute to the fish kills. We hypothesized that prior exposure to adverse water quality conditions increases the susceptibility of Lost River suckers to F. columnare infections. To test this, we exposed juvenile Lost River suckers to four sublethal ammonia concentrations at pH 9.4 for 62 d. On day 31, fish in half of the aquaria were exposed to F. columnare. As expected, survival of the Lost River suckers decreased in aquaria inoculated with F. columnare. Ninety-four percent of the fish that died were infected by F. columnare in the gills, kidney, or skin, whereas none of the survivors or unexposed control fish was infected. However, contrary to our hypothesis, survival of the fish exposed to F. columnare increased significantly (p < 0.05) as unionized ammonia concentrations increased. Our results suggest that complex interactions can complicate prediction of the responses of fish to concurrent chemical stressors and bacterial pathogens.