Assessing the accuracy of a polymerase chain reaction test for Ichthyophonus hoferi in Yukon River Chinook salmon Oncorhynchus tshawytscha

Ichthyophonus hoferi Plehn & Mulsow, 1911, is a cosmopolitan, protistan pathogen of marine fishes. It is prevalent in mature returning Chinook salmon Oncorhynchus tshawytscha in the Yukon River watershed, and may be associated with prespawning mortality. We developed and evaluated a polymerase chain reaction (PCR) test for I. hoferi using primers specific to the parasite's small subunit rDNA. The test has a minimum detection limit of approximately 10(-5) parasite spores per reaction and does not cross-react with the closely related salmon parasites Dermocystidium salmonis or Sphaerothecum destruens. Sensitivity and specificity of the PCR test used on somatic muscle and heart tissue for detecting infected fish were determined using 334 Chinook salmon collected from the Yukon River at 2 locations (Tanana and Emmonak) in 2003 and 2004. The true infection status of the fish was determined by testing somatic muscle, heart and kidney tissue using histological evaluation, culture, and PCR. The severity of infection was grouped into 2 categories, light and heavy infection. The probability of detecting a heavily infected fish (sensitivity of the test) was generally much higher than the probability of detecting light infection, suggesting that more than one tissue and/or method should be used to accurately detect light or early infection by I. hoferi. The probability of correctly identifying a negative fish (specificity of the test) was always greater than 94% regardless of the tissue used, infection severity, sampling site or year of collection.     

Introduced pathogens and native freshwater biodiversity: a case study of Sphaerothecum destruens

A recent threat to European fish diversity was attributed to the association between an intracellular parasite, Sphaerothecum destruens, and a healthy freshwater fish carrier, the invasive Pseudorasbora parva originating from China. The pathogen was found to be responsible for the decline and local extinction of the European endangered cyprinid Leucaspius delineatus and high mortalities in stocks of Chinook and Atlantic salmon in the USA. Here, we show that the emerging S. destruens is also a threat to a wider range of freshwater fish than originally suspected such as bream, common carp, and roach. This is a true generalist as an analysis of susceptible hosts shows that S. destruens is not limited to a phylogenetically narrow host spectrum. This disease agent is a threat to fish biodiversity as it can amplify within multiple hosts and cause high mortalities.     

Aquatic Francisella-like bacterium associated with mortality of intensively cultured hybrid striped bass Morone chrysops x M. saxatilis

The present study identifies an emerging disease associated with an aquatic Francisella-like bacterium that can cause mortality in hybrid striped bass Morone chrysops x M. saxatilis reared intensively in freshwater. Clinically affected fish were lethargic, had scattered haemorrhagic cutaneous lesions and diffuse gill pallor. The head kidney and spleen were markedly swollen and contained numerous interstitial granulomas; histological examination revealed small, pleomorphic Gram-negative coccobacilli within vacuolated cells. The bacterium could not be cultured from head kidney homogenates either with standard or enriched microbiological media or following inoculation of a Chinook salmon embryo (CHSE)-214 cell line. No amplification product was obtained from head kidney DNA by polymerase chain reaction (PCR) assay using Piscirickettsia salmonis-specific primers. PCR analysis of infected head kidney homogenate with primers designed for the eubacterial 16S rRNA produced a single amplicon. Phylogenetic analysis of this DNA sequence demonstrated that the sequence aligned most closely with members of the genus Francisella, identified from tilapia Oreochromis spp. in Taiwan and an aquatic Francisella species that was recently isolated from the three-line grunt Parapristipoma trilineatum in Japan. This Francisella-like disease was transmitted to naive hybrid striped bass fingerlings by intraperitoneal injection of tissue homogenates prepared from a natural outbreak. All fish developed gross and histological lesions identical to those from natural outbreaks. Intracellular Gram-negative bacteria were observed within the cytoplasm of cells (presumably macrophages) within the granulomas, but bacteria were not recovered. The 16S DNA sequence of the bacterium obtained from tissues of experimentally infected fish was identical to that obtained from the fish used as infected donor tissue.     

Invasion of Ceratomyxa shasta (Myxozoa) and comparison of migration to the intestine between susceptible and resistant fish hosts

The myxozoan parasite Ceratomyxa shasta infects salmonids causing ceratomyxosis, a disease elicited by proliferation of the parasite in the intestine. This parasite is endemic to the Pacific Northwest of North America and salmon and trout strains from endemic river basins show increased resistance to the parasite. It has been suggested that these resistant fish (i) exclude the parasite at the site of invasion and/or (ii) prevent establishment in the intestine. Using parasites pre-labeled with a fluorescent stain, carboxyfluorescein succinimidyl diacetate (CFSE), the gills were identified as the site of attachment of C. shasta in a susceptible fish strain. In situ hybridization (ISH) of histological sections was then used to describe the invasion of the parasites in the gill filaments. To investigate differences in the progress of infection between resistant and susceptible fish, a C. shasta-susceptible strain of rainbow trout (Oncorhynchus mykiss) and a C. shasta-resistant strain of Chinook salmon (Oncorhynchus tshawytscha) were sampled at consecutive time points following exposure at an endemic site. Using ISH in both species, the parasite was observed to migrate from the gill epithelium into the gill blood vessels where replication and release of parasite stages occurred. Quantitative PCR verified entry of the parasite into the blood. Parasite levels in blood increased 4 days p.i. and remained at a consistent level until the second week when parasite abundance increased further and coincided with host mortality. The timing of parasite replication and migration to the intestine were similar for both fish species. The field exposure dose was unexpectedly high and apparently overwhelmed the Chinook salmon’s defenses, as no evidence of resistance to parasite penetration into the gills or prevention of parasite establishment in the intestine was observed.     

Development and application of a nested PCR to monitor brood stock salmonid ovarian fluid and spleen for detection of the fish pathogen Flavobacterium psychrophilum

AIMS: To develop a nested PCR to detect Flavobacterium psychrophilum based on the intergenic spacer region 16S-23S rRNA and in 16S rRNA for analysis of brood stock salmonid fish samples. METHODS AND RESULTS: The sensitivity and specificity of the test was evaluated using pure cultures, spiked and naturally contaminated samples. Samples were internal organs (spleen and kidney), eggs and ovarian fluid from rainbow trout and coho salmon from European fish farms (France, Spain). This nested PCR was more specific and sensitive that the nested PCR based on 16S rRNA sequences primers only. The detection limit of this PCR assay was one bacterium per PCR tube corresponding to 10 bacteria/mg of spleen and 5 bacteria/ml from ovarian fluid. Analysis of mixed ovarian fluid samples from reproductive salmonids in various French hatcheries demonstrated that 69% of hatcheries were contaminated with Fl. psychrophilum. The analysis of individual samples demonstrated that 39% of rainbow trout (Oncorhynchus mykiss) and 62.5% of coho salmon (O. kisutch) samples were contaminated. CONCLUSIONS: The results demonstrated a very sensitive and specific detection of this fish pathogen and that most of the female rainbow trout and coho salmon breeders analysed carry Fl. psychrophilum in the ovarian fluid. SIGNIFICANCE AND IMPACT OF THE STUDY: The understanding of Fl. psychrophilum dissemination and transmission and the detection of asymptomatic carriers is important for the development of free breeders stock and for significantly decreasing Flavobacteriose.     

Efficacy of an infectious hematopoietic necrosis (IHN) virus DNA vaccine in Chinook Oncorhynchus tshawytscha and sockeye O. nerka salmon

The level of protective immunity was determined for Chinook Oncorhynchus tshawytscha and sockeye/kokanee salmon (anadromous and landlocked) O. nerka following intramuscular vaccination with a DNA vaccine against the aquatic rhabdovirus, infectious hematopoietic necrosis virus (IHNV). A DNA vaccine containing the glycoprotein gene of IHNV protected Chinook and sockeye/kokanee salmon against waterborne or injection challenge with IHNV, and relative percent survival (RPS) values of 23 to 86% were obtained under a variety of lethal challenge conditions. Although this is significant protection, it is less than RPS values obtained in previous studies with rainbow trout (O. mykiss). In addition to the variability in the severity of the challenge and inherent host susceptibility differences, it appears that use of a cross-genogroup challenge virus strain may lead to reduced efficacy of the DNA vaccine. Neutralizing antibody titers were detected in both Chinook and sockeye that had been vaccinated with 1.0 and 0.1 pg doses of the DNA vaccine, and vaccinated fish responded to viral challenges with higher antibody titers than mock-vaccinated control fish.     

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.     

Class I MHC polymorphism and evolution in endangered California Chinook and other Pacific salmon

Twelve MHC class I exon 2 sequences were uncovered in a sample from the endangered Sacramento River winter-run Chinook salmon in the central valley of California. Phylogenetic analysis of the 12 sequences indicates that the alleles descend from two of six major allelic lineages found among four Pacific salmon species. Nine of the 12 alleles belong to an allelic lineage that began diversifying 8 million years ago, just prior to the estimated time of Chinook speciation. The most recent common ancestor of all 12 winter-run alleles is estimated to be 15 million years ago, approximately 5 million years before the radiation of the Pacific salmon species. The average nonsynonymous distance among the peptide binding-region codons of exon 2 for the 12 alleles is significantly higher than the average synonymous distance in these codons. We estimate the symmetrical overdominant selection coefficient against homozygotes for this exon to be 0.038. Thus, strong positive and balancing selection has maintained functional diversity in the peptide-binding region of the exon over millions of years and this variation has not yet been substantially eliminated by increased genetic drift due to the recent dramatic decline in abundance of this Chinook salmon population.     

Susceptibility of captive adult winter-run Chinook salmon Oncorhynchus tshawytscha to waterborne exposures with infectious hematopoietic necrosis virus (IHNV)

Sexually mature female Chinook salmon Oncorhynchus tshawytscha with no prior history of exposure to infectious hematopoietic necrosis virus (IHNV) were susceptible to experimental infection induced by additions of virus to the water. The resulting infections resembled those observed among naturally infected hatchery and wild populations of Chinook salmon. Virus was detected as early as 4 d post-exposure (p.e.) and subsequently in all virus-exposed fish that died or that were examined at 14 d p.e. when the study was terminated. The greatest concentrations of virus, up to 10(8) plaque-forming units (pfu) ml(-1), were found in the ovarian fluid at 13 to 14 d p.e., but the virus was also found in high concentrations in the gill, kidney/spleen and plasma. In contrast, the virus was not recovered from unexposed control adult salmon that died or were sampled at the end of the study. Despite detecting concentrations of IHNV in excess of 10(7) pfu g(-1) of tissue, no specific microscopic lesions were found in IHNV-exposed compared to unexposed control salmon. The results of this initial study suggest that virus in the spawning environment, either from adult salmon or other sources, may contribute to its rapid spread among adult Chinook salmon, thereby considerably increasing the prevalence of IHNV infection in both wild and hatchery populations of adult Chinook salmon.     

SSU rRNA gene sequence reveals two genotypes of Spironucleus barkhanus (Diplomonadida) from farmed and wild Arctic charr Salvelinus alpinus

Spironucleus barkhanus isolated from the blood of Arctic charr Salvelinus alpinus from a marine fish farm were genetically compared with S. barkhanus isolated from the gall bladder of wild Arctic charr. The wild Arctic charr were caught in the lake used as the water source for the hatchery from which the farmed fish originated. Sequencing of the small subunit ribosomal RNA gene (SSU rDNA) from these 2 populations showed that the isolates obtained from farmed and wild Arctic charr were only 92.7 % similar. Based on the sequence differences between these isolates, it is concluded that the parasites isolated from the farmed fish have not been transmitted from wild Arctic charr in the hatchery's fresh water source. It is therefore most likely that the farmed fish were infected by S. barkhanus after they were transferred to seawater. S. barkhanus isolated from diseased farmed Arctic charr were 99.7% similar to the isolates obtained from diseased farmed Chinook (Canada) and Atlantic salmon (Norway). The high degree of sequence similarity between S. barkhanus from farmed Arctic charr, Chinook and Atlantic salmon indicates that systemic spironucleosis may be caused by specific strains/variants of this parasite. The genetic differences between the isolates of farmed and wild fish are of such magnitude that their conspecificity should be questioned.     

Isolation and culture of Sphaerothecum destruens from Sunbleak (Leucaspius delineatus) in the UK and pathogenicity experiments in Atlantic salmon (Salmo salar)

The sunbleak (Leucaspius delineatus), a cyprinid fish native to continental Europe and now established in the UK, is experiencing population decline which appears to be linked to the spread of the invasive Asian cyprinid (Pseudorasbora parva). A population of sunbleak in the UK has previously been identified as infected with S. destruens at low prevalence. Because Sphaerothaecum destruens has, on occasion, caused severe disease in cultured and wild salmonids the aim of this work was to establish laboratory cultures of S. destruens from sunbleak in the UK and use these cultures in challenge experiments to determine if the UK isolate of S. destruens from cyprinid species is a potential threat to Atlantic salmon (Salmo salar). The first isolation and culture of S. destruens in the UK and from a cyprinid species is described. Cultured S. destruens spores from sunbleak are infective to EPC, CHSE and FHM cells, replicating most rapidly in FHM and EPC cells. Spores can be induced to zoosporulate in water forming motile, uni-flagellated zoospores. Challenge experiments indicated the spores are able to replicate and disperse in Atlantic salmon and are associated with increased mortality (up to 90%) when injected intraperitonealy.     

Epitopes associated with mature spores not recognized on Kudoa thyrsites from recently infected Atlantic salmon smolts

Atlantic salmon Salmo salar skeletal muscle was examined for Kudoa thyrsites by polymerase chain reaction (PCR) and positive fish were further examined by in situ hybridization (ISH) and immunohistochemistry (IHC). The infection was detected in 42% of salmon by PCR following a 60 d exposure to infective seawater at a temperature of 10 degrees C (= 600 degree-days, degreeD). The parasite was detected by ISH in skeletal and cardiac muscle but not in gill, kidney, spleen, liver, stomach, intestine, pyloric caeca and skin. None of 4 monoclonal antibodies (2F4, 4H2, 1H2, 3E8) raised against mature K. thyrsites spores reacted with the stages identified by ISH following a 600 degreeD exposure, but they did react with ISH-identified stages following a 1600 degreeD exposure. In contrast, a polyclonal antibody reacted with K. thyrsites stages in salmon with both 600 and 1600 degreeD exposures, suggesting that the parasite observed in 600 degreeD infections represents an antigenically distinct developmental stage of K. thyrsites.     

Experimental and natural host specificity of Loma salmonae (Microsporidia)

The microsporidian Loma salmonae (Putz, Hoffman & Dunbar, 1965) Morrison & Sprague, 1981 has caused significant gill disease in Pacific salmon Oncorhynchus spp. Host specificity of the parasite was examined experimentally by per os challenge of selected salmonids and non-salmonids with infective chinook salmon O. tshawytscha gill material. Pink Oncorhynchus gorbuscha and chum salmon O. keta, brown Salmo trutta and brook trout Salvelinus fontinalis, and chinook salmon (controls) were positive, whereas Atlantic salmon Salmo salar and Arctic char Salvelinus alpinus were negative. In addition, no non-salmonids were susceptible to experimental exposure. Wild Pacific salmon species in British Columbia, Canada, were examined for L. salmonae during their freshwater life history stages (smolts, prespawning, spawning). All stages were infected, although infections in smolts were only detectable using a L. salmonae-specific PCR test. Many previous Loma spp. described from Oncorhychus spp. are likely L. salmonae based on host, parasite morphology, and site of infection.     

Interpreting dual ELISA and qPCR data for bacterial kidney disease of salmonids

Although there are a variety of methods available for the detection of Renibacterium salmoninarum, the causative agent of bacterial kidney disease in salmon and trout, the enzyme-linked immunosorbent assay (ELISA) is probably the most widely used method. However, ELISA measures bacterial antigen, which does not necessarily reflect the number of cells present. We hypothesized that dual analysis of kidney tissue by ELISA and a quantitative real-time polymerase chain reaction assay (qPCR) would provide complementary information about antigen level and the number of bacterial genomes. We found that DNA extracted from the insoluble fraction of the ELISA tissue preparation produced the same qPCR result as DNA extracted directly from frozen tissue, permitting true dual analysis of the same tissue sample. We examined kidney tissue in this manner from individual free-ranging juvenile Chinook salmon and antibiotic-treated captive subadult Chinook salmon and observed 3 different patterns of results. Among the majority of fish, there was a strong correlation between the ELISA value and the qPCR value. However, subsets of fish exhibited either low ELISA values with elevated qPCR values or higher ELISA values with very low qPCR values. These observations suggest a conceptual model that allows inferences about the state of infection of individual fish based on dual ELISA/qPCR results. Although this model requires further assessment through experimental infections and treatments, it may have utility in broodstock selection programs that currently apply egg-culling practices based on ELISA alone.     

Involvement of Manayunkia speciosa (Annelida: Polychaeta: Sabellidae) in the life cycle of Parvicapsula minibicornis, a myxozoan parasite of Pacific salmon

A coelomic myxozoan infection was detected in freshwater polychaetes, Manayunkia speciosa from the Klamath River, Oregon/California, a site enzootic for the myxozoan parasites Ceratomyxa shasta and Parvicapsula minibicornis. The tetractinomyxon type actinospores had a near-spherical spore body 7.9 x 7.1 microm, with 3 spherical, protruding polar capsules, no valve cell processes, and a binucleate sporoplasm. Parvicapsula minibicornis-specific primers Parvi1f and Parvi2r amplified DNA from infected polychaetes in a polymerase chain reaction (PCR) assay. The small subunit 18S rRNA gene of the spores was sequenced (GenBank DQ231038) and was a 99.7% match with the sequence for P. minibicornis myxospore stage in GenBank (AF201375). Chinook salmon (Oncorhynchus tshawytscha) exposed to a dose of 1,000 actinospores per fish tested PCR positive for P. minibicornis at 14 wk postinfection and presporogonic stages were detected in the kidney tubules by histology at 20 wk. This life cycle is 1 of only about 30 known from more than 1,350 myxozoan species, and only the second known from a freshwater polychaete.     

Survey of parasites in threatened stocks of coho salmon (Oncorhynchus kisutch) in Oregon by examination of wet tissues and histology

We are conducting studies on the impacts of parasites on Oregon coastal coho salmon (Oncorhynchus kistuch). An essential first step is documenting the geographic distribution of infections, which may be accomplished by using different methods for parasite detection. Thus, the objectives of the current study were to (1) identify parasite species infecting these stocks of coho salmon and document their prevalence, density, and geographic distribution; (2) assess the pathology of these infections; and (3) for the first time, determine the sensitivity and specificity of histology for detecting parasites compared with examining wet preparations for muscle and gill infections. We examined 576 fry, parr, and smolt coho salmon in total by histology. The muscle and gills of 219 of these fish also were examined by wet preparation. Fish were collected from 10 different locations in 2006-2007. We identified 21 different species of parasites in these fish. Some parasites, such as Nanophyetus salmincola and Myxobolus insidiosus, were common across all fish life stages from most basins. Other parasites, such as Apophallus sp., were more common in underyearling fish than smolts and had a more restricted geographic distribution. Additional parasites commonly observed were as follows: Sanguinicola sp., Trichodina truttae , Epistylis sp., Capriniana piscium, and unidentified metacercariae in gills; Myxobolus sp. in brain; Myxidium salvelini and Chloromyxum majori in kidney; Pseudocapillaria salvelini and adult digenean spp. in the intestine. Only a few parasites, such as the unidentified gill metacercariae, elicted overt pathologic changes. Histology had generally poor sensitivity for detecting parasites; however, it had relatively good specificity. We recommend using both methods for studies or monitoring programs requiring a comprehensive assessment of parasite identification, enumeration, and parasite-related pathology.     

Resistance to reinfection in chinook salmon Oncorhynchus tshawytscha to Loma salmonae (Microsporidia).

Chinook salmon Oncorhynchus tshawytscha were experimentally infected per os with Loma salmonae and held in flow-through seawater tanks at 12 to 14 degrees C. The fish exhibited 100% infection when first examined at 7 wk post initial exposure (p.e.), and by 20 wk p.e. they had completely recovered from gill infections. The recovered fish were then re-exposed the following week. All of these fish showed strong protection to new L. salmonae infections, while na?ve fish exposed to the same inoculum developed the infection. Most of the re-exposed fish exhibited a few free spores or spores within phagocytes in the kidney interstitium at 20 to 29 wk p.e., but xenomas were not detected in either the gills or visceral organs. The kidney is the primary site of reticulo-endothelial activity, and thus these spores were likely deposited in the kidney by entrapment by fixed macrophages. It is possible that these spores provide immunologic stimuli to reinforce the resistance to new L. salmonae infections.