Salmon poisoning disease in juvenile coyotes: clinical evaluation and infectivity of metacercariae and rickettsiae

Clinical salmon poisoning disease (SPD), and survival of Neorickettsia helminthoeca and metacercariae of Nanophyetus salmincola in fish were evaluated experimentally in 12-wk-old coyotes (Canis latrans) to determine the potential of SPD for biological control of coyotes. Coyotes readily ate fish that contained metacercariae and rickettsiae. They developed diarrhea, anorexia and lethargy within 7 days after feeding. Infected coyotes lost 58% of their body weight when compared to uninfected controls. They died or became moribund and were euthanatized within 17 days after feeding. Rickettsiae were present in the macrophages of lymph nodes of all affected coyotes. Clinical disease occurred in coyotes fed fresh fish, but not in coyotes fed fish stored at 4 C for greater than or equal to 30 days or at -20 C for 14 days. Metacercariae in fish were viable after 60 days at 4 C. These trematodes developed in coyotes, but clinical SPD did not occur. This indicated survival of metacercariae, but not rickettsiae. Metacercariae were not viable after 14 days at -20 C.     

Isolation and characterization of Edwardsiella tarda from fall chinook salmon (Oncorhynchus tshawytscha).

A new bacterial pathogen of chinook salmon (oncorhynchus tshawytscha) was isolated from fish in Oregon's Rogue River. The bacteria are biochemically and serologically related to strains of Edwardsiella tarda. Initially isolated from chinook salmon, the bacteria were also pathogenic for steelhead and rainbow trout (Salmo gairdneri), and channel catfish (Ictalurus punctatus). The 50% lethal doses for chinook salmon, steelhead trout, and channel catfish injected intraperitoneally and maintained in 18 degrees C water were 4.1 x 10(6), 5.6 x 10(6), and 4.0 x 10(5) respectively. When chinook salmon and rainbow trout were injected intraperitoneally and held in 12 degrees C water, the mean lethal doses were 6.4 x 10(7) and 1.7 x 10(6), respectively. The invasiveness of the organism was low in steelhead trout exposed to the bacteria by the waterborne route. The optimum growth temperature of the bacteria in brain heart infusion broth was approximately 35 degrees C. The guanine plus cytosine content of DNA obtained from E. tarda isolated from salmon was 59 mol%.     

Vulnerability to predation and physiological stress responses of experimentally descaled juvenile chinook salmon,Oncorhynchus tshawytscha

Synopsis Juvenile salmonids,Oncorhynchus spp., commonly encounter conditions (e.g., during hatchery release and dam passage) that result in damage to the skin, scale, and slime complex. We conducted laboratory experiments to determine if descaling of juvenile chinook salmon,O. tshawytscha, increased their vulnerability to predation, and to assess the physiological stress responses elicited by descaling. Salmon were experimentally descaled on either 10% or 20% of their total body area. When offered equal numbers of control and descaled juvenile chinook salmon, northern squawfish,Ptychocheilus oregonensis, did not consume significantly more of either prey type (48–60% of consumed prey were descaled). Juvenile chinook salmon descaled on 10% of their body area did show significant physiological stress responses, however. Mean concentrations of plasma cortisol peaked 1 h after descaling, and returned to control levels by 12 h. Plasma glucose peaked 3 h post-treatment and remained elevated for 24 h. Plasma lactate increased immediately following treatment and returned to undisturbed control levels by 3 h. The osmoregulatory response of plasma potassium was highly variable, but plasma sodium decreased immediately and remained low for 24 h. The observed physiological responses suggest that descaling of juvenile chinook salmon could result in decreased resistance to disease and other stressors encountered in the field, possibly leading to reduced performance capacity and lowered survival.     

Xenoma formation during microsporidial gill disease of salmonids caused by Loma salmonae is affected by host species (Oncorhynchus tshawytscha,O. kisutch,O. mykiss) but not by salinity

Host species and salinity often affect the development of disease in aquatic species. Eighty chinook salmon Oncorhynchus tshawytscha, 80 coho salmon O. kisutch and 80 rainbow trout O. mykiss were infected with Loma salmonae. Forty of each species were reared in seawater and 40 in freshwater. The mean number of xenomas per gill filament was 8 to 33 times greater in chinook salmon than in rainbow trout (RBT). Coho salmon had a mean xenoma intensity intermediate to that of chinook salmon and RBT. In contrast to the differences between species, salinity had no significant effect on xenoma intensity in any of these host species. The onset of xenoma formation occurred at Week 5 postexposure (PE) for chinook salmon and RBT, and at Week 6 PE for coho salmon. RBT had cleared all visible branchial xenomas by Week 9 PE, whereas xenomas persisted in coho and chinook salmon at Week 9 PE. Histologically, xenomas were visible in the filament arteries of the branchial arch in chinook and coho salmon gills but were absent from RBT gills. Fewer xenomas were seen in the central venous sinusoids of RBT than in chinook and coho salmon. The lower xenoma intensity, shorter duration of infection and pathological characteristics, common to microsporidial gill disease in RBT, suggest a degree of resistance to clinical disease that is not seen in coho and chinook salmon.     

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.     

Glochidiosis of salmonid fishes. II. Comparison of tissue response of coho and chinook salmon to experimental infection with Margaritifera margaritifera (L.) (Pelecypoda: Margaritanidae)

Coho salmon (Oncorhynchus kisutch) are more resistant to experimental infection with the glochidia of the freshwater mussel (Margaritifera margaritifera) than are chinook salmon (Oncorhynchus tshawytscha). Histological sections made at intervals during the infection showed that coho salmon sloughed the parasites from their gills by 4.5 days postinfection, but the parasites remained encysted in the gills of chinook salmon for 12 weeks, when metamorphosis to juvenile mussels was complete. Coho salmon sloughed the parasites by a well-developed hyperplasia. No such pronounced hyperplastic reaction was seen in the gills of chinook salmon.     

The road to extinction is paved with good intentions: negative association of fish hatcheries with threatened salmon

Hatchery programmes for supplementing depleted populations of fish are undergoing a worldwide expansion and have provoked concern about their ramifications for populations of wild fish. In particular, Pacific salmon are artificially propagated in enormous numbers in order to compensate for numerous human insults to their populations, yet the ecological impacts of this massive hatchery effort are poorly understood. Here we test the hypothesis that massive numbers of hatchery-raised chinook salmon reduce the marine survival of wild Snake River spring chinook, a threatened species in the USA. Based on a unique 25-year time-series, we demonstrated a strong, negative relationship between the survival of chinook salmon and the number of hatchery fish released, particularly during years of poor ocean conditions. Our results suggest that hatchery programmes that produce increasingly higher numbers of fish may hinder the recovery of depleted wild populations.     

Evidence for a carrier state of infectious hematopoietic necrosis virus in chinook salmon Oncorhynchus tshawytscha.

In British Columbia, Canada, infectious hematopoietic necrosis virus (IHNV) is prevalent in wild sockeye salmon Oncorhynchus nerka and has caused disease in seawater net-pen reared Atlantic salmon Salmo salar. In this study, chinook salmon Oncorhynchus tshawytscha experimentally exposed to an isolate of IHNV found in British Columbia became carriers of the virus. When Atlantic salmon were cohabited with these virus-exposed chinook salmon, IHNV was isolated from the Atlantic salmon. Identification of chinook salmon populations that have been exposed to IHNV may be difficult, as virus isolation was successful only in fish that were concurrently infected with either Renibacterium salmoninarum or Piscirickettisia salmonis. Also, IHNV-specific antibodies were detected in only 2 of the 70 fish experimentally exposed to the virus. Two samples collected from chinook salmon exposed to IHNV while at a salt water net-pen site had a seroprevalence of 19 and 22%; however, the inconsistencies between our laboratory and field data suggest that further research is required before we can rely on serological analysis for identifying potential carrier populations. Because of the difficulty in determining the exposure status of populations of chinook salmon, especially if there is no concurrent disease, it may be prudent not to cohabit Atlantic salmon with chinook salmon on a farm if there is any possibility that the latter have been exposed to the virus.     

Effects of natural selection on patterns of DNA sequence variation at the transferrin, somatolactin, and p53 genes within and among chinook salmon (Oncorhynchus tshawytscha) populations

This paper describes DNA sequence variation within and among four populations of chinook salmon (Oncorhynchus tshawytscha) at the transferrin, somatolactin and p53 genes. Patterns of variation among salmon species at the transferrin gene have been hypothesized to be shaped by positive natural selection for new alleles because the rate of nonsynonymous substitution is significantly greater than the rate of synonymous substitution. The twin goals of this study were to determine if the history of selection among salmon species at the transferrin gene is also reflected in patterns of intraspecific variation in chinook salmon, and to look for evidence of local adaptation at the transferrin gene by comparing patterns of nonsynonymous and synonymous variation among chinook salmon populations. The analyses presented here show that unlike patterns of variation between species, there is no evidence of greater differentiation among chinook salmon populations at nonsynonymous compared to synonymous sites. There is also no evidence of a reduction of within-species variation due to the hitchhiking effect at the transferrin gene, although in some populations nonsynonymous and synonymous derived mutations are both at higher frequencies than expected under a simple neutral model. Population size weighted selection coefficients (4Ns) that are consistent with both the inter and intraspecific data range from approximately 10 to approximately 235, and imply that between 1 and 40% of new nonsynonymous mutations at the transferrin gene have been beneficial.     

Different prevalences of Renibacterium salmoninarum detected by ELISA in Alaskan chinook salmon Oncorhynchus tshawytscha spawned from freshwater and seawater

Soluble antigen of Renibacterium salmoninarum (Rs) was detected by a polyclonal enzyme-linked immunosorbent assay (ELISA) at significantly higher prevalences in adult chinook salmon Oncorhynchus tshawytscha that matured in freshwater than in the same cohort of fish spawned after maturation in seawater. The cumulative results were consistent during 4 yr of comparison at the Little Port Walter Hatchery on Baranof Island, Alaska, USA. Possible causes for this difference are discussed. Maturation of chinook salmon broodstock in seawater has become a practical strategy at this hatchery to reduce the prevalence of Rs-positive parent fish and the numbers of culled eggs.     

Identification of the sex chromosome pair in coho salmon (Oncorhynchus kisutch): lack of conservation of the sex linkage group with chinook salmon (Oncorhynchus tshawytscha)

Fluorescence in situ hybridization (FISH) using a probe to the male-specific GH-Y (growth hormone pseudogene) was used to identify the Y chromosome in coho salmon (Oncorhynchus kisutch). The sex chromosome pair is morphologically similar to chinook salmon (Oncorhynchus tshawytscha) with the GH-Y localized to the small short arm of the largest subtelocentric chromosome pair. FISH experiments with probes containing sex-linked genes in rainbow trout (Oncorhynchus mykiss) (SCAR163) and chinook salmon (Omy7INRA) showed that the coho sex linkage group is different from chinook and rainbow trout and this was confirmed by segregation analysis for the Omy7INRA locus. The telomeric location of the SEX locus, the presence of shared male-specific markers in coho and chinook salmon, and the lack of conservation of sex-linkage groups suggest that transposition of a small male-specific region may have occurred repeatedly in salmonid fishes of the genus Oncorhynchus.     

In vitro detection of functional humoral immunocompetence in juvenile chinook salmon (Oncorhynchus tshawytscha) using flow cytometry

A flow cytometric (FCM) assay for detection of immunomodulatory effects of environmental factors on the humoral response of chinook salmon (Oncorhynchus tshawytscha) is described and validated. This technique combines exposure of whole animals or leucocyte cultures to immunomodulatory agents/conditions with in vitro mitogenic activation of B-lymphocytes. The proportion of leucocytes undergoing blastogenesis following in vitro stimulation with lipopolysaccharide (LPS) is quantified by FCM analysis of forward and side scatter properties. In addition, binding of a fluorescein isothiocyanate labelled anti-rainbow trout immunoglobulin M monoclonal antibody (anti-RBT SIgM-FITC), quantified by FCM analysis, is used to determine the ability of the lymphoblasts to express surface immunoglobulin M (SIgM).Through a series of calibration steps, it was confirmed that anti-RBT IgM-FITC was specific for B-lymphocyte SIgM in chinook salmon. Binding of anti-RBT IgM-FITC to chinook salmon SIgM positive leucocytes was effectively blocked with salmon serum and an isotype control was established. B-lymphocytes were partially removed from a population of leucocytes through adherence to a nylon wool column, which then demonstrated a consequent reduction in anti-RBT IgM-FITC binding. Using anti-RBT IgM-FITC as a marker, the distribution of resting lymphocytes expressing SIgM in lymphoid tissues of juvenile chinook salmon was described. The mean percentage of SIgM positive cells in spleen, pronephros and blood were found to be 62.1 (+/-2.82), 34.8 (+/-1.86) and 56.7% (+/-4.7) of all viable leucocytes, respectively. In a time-course experiment for optimal in vitro activation of leucocytes for this assay, blastogenesis and up-regulation of SIgM expression of splenic leucocytes were observed through FCM by 4 days post in vitro stimulation with LPS, continued through 7 days, but was no longer visible by 10 days post stimulation. Using this assay, reduced expression of SIgM in splenic and pronephric B-lymphocytes was detected following in vitro exposure to physiologically relevant stress concentrations of cortisol in conjunction with mitogenic stimulation. This technique will be a useful addition to the assays already available in the rapidly growing field of fish immunology.     

The aunt and uncle effect: an empirical evaluation of the confounding influence of full sibs of parents on pedigree reconstruction.

This study used simulations and a known two-generation pedigree of chinook salmon (Oncorhynchus tshawytscha) to evaluate the effect of full sibs of parents on pedigree reconstruction. Parentage analysis was conducted on 100 parent pair-offspring relationships from pedigrees with unrelated (simulation) and related (chinook salmon) candidate parents. Parentage assignment success for the chinook salmon was lower than in the simulated populations. For example, the six most variable loci (mean H(E) = 0.87) provided a mean of 97% unambiguous assignments in the simulated population and 67% unambiguous assignments for the chinook salmon. Estimates of the pairwise relatedness coefficient ((xy)) for most nonexcluded false parents and true parents of chinook salmon offspring exceeded 0.50. These results support the conclusion that closely related candidate parents decrease the power of genetic markers for pedigree reconstruction based on exclusion. Ambiguous parentage may be resolved using single parent- and parent pair-offspring likelihood analysis, however, these methods should be used with caution and they are not replacements for using more loci when many candidate parents are full sibs.     

Behavioral Thermoregulation by Juvenile Spring and Fall Chinook Salmon, Oncorhynchus Tshawytscha, during Smoltification

Fall chinook salmon evolved to emigrate during the summer months. The shift in the temperature preference we observed in smolting fall chinook but not spring chinook salmon may reflect a phylogenetic adaptation to summer emigration by (1) providing directional orientation as fall chinook salmon move into the marine environment, (2) maintaining optimal gill function during emigration and seawater entry, and/or (3) resetting thermoregulatory set-points to support physiological homeostasis once smolted fish enter the marine environment. Phylogenetically determined temperature adaptations and responses to thermal stress may not protect fall chinook salmon from the recent higher summer water temperatures, altered annual thermal regimes, and degraded cold water refugia that result from hydropower regulation of the Columbia and Snake rivers. The long-term survival of fall chinook salmon will likely require restoration of normal annual thermographs and rigorous changes in land use practices to protect critical thermal refugia and control maximum summer water temperatures in reservoirs.     

Pseudobranch and gill Na(+), K(+)-ATPase activity in juvenile chinook salmon,Oncorhynchus tshawytscha: developmental changes and effects of growth hormone,cortisol and seawater transfer

The teleost pseudobranch is a gill-like structure often fused to the anterior of the opercular cavity. Pseudobranch cells are mitochondria rich and have high levels of Na(+), K(+)-ATPase activity. In this study, pseudobranch Na(+), K(+)-ATPase activity in juvenile chinook salmon (Oncorhynchus tshawytscha) was compared to gill Na(+), K(+)-ATPase activity, a known marker of parr-smolt transformation, in three experiments. In two stocks of New Zealand chinook salmon, pseudobranch Na(+), K(+)-ATPase activity was found to significantly increase during development. At these times gill Na(+), K(+)-ATPase activity was also elevated. Pseudobranch Na(+), K(+)-ATPase activity did not increase 10 days after transfer from fresh water to 34 ppt seawater, a treatment that resulted in a twofold increase in gill Na(+), K(+)-ATPase activity. Cortisol (50 microg/g) and ovine growth hormone (5 microg/g) implants had no effect on pseudobranch Na(+), K(+)-ATPase activity in underyearling chinook salmon, while gill Na(+), K(+)-ATPase activity was stimulated by each hormone. In yearling chinook salmon, only cortisol stimulated pseudobranch Na(+), K(+)-ATPase activity 14 days post-implantation. It was concluded that the pseudobranch differs from the gill in terms of the regulation of Na(+), K(+)-ATPase activity and a role during adaptation to seawater is likely to be limited.     

In Vitro Characteristics of the Microsporidian: Enterocytozoon salmonis

ABSTRACT. Enterocytozoon salmonis , an intranuclear microsporidian of salmonid fish, was propagated in vitro using chinook salmon mononuclear leukocytes. Characteristic morphology and infectivity of the cultured parasites were evaluated to determine the effect of in vitro maintenance and passage on the parasites. Cultured parasites developed through several stages from meronts to infectious spores. Parasites obtained from in vitro passages tested up to the 17th subculture, retained their morphological characteristics and pathogenicity for chinook salmon ( Oncorhynchus tshawytscha) . The disease induced by experimental infections with parasites from in vitro cultures was ideniical to that observed in naturally infected chinook salmon. An examination of supernatants obtained from the infected cultures revealed evidence of soluble factor(s) produced by E. salmonis -infected cells that stimulated uninfected target cells in vitro. This observation may explain in part the proliferative disease of hematopoietic tissues which characterizes the disease in infected chinook salmon.     

Isolation and characterization of a trypsin fraction from the pyloric ceca of chinook salmon (Oncorhynchus tshawytscha)

A trypsin fraction was isolated from the pyloric ceca of New Zealand farmed chinook salmon (Oncorhynchus tshawytscha) by ammonium sulfate fractionation, acetone precipitation and affinity chromatography. The chinook salmon enzyme hydrolyzed the trypsin-specific synthetic substrate benzoyl-dl-arginine-p-nitroanilide (dl-BAPNA), and was inhibited by the general serine protease inhibitor phenyl methyl sulfonyl fluoride (PMSF), and also by the specific trypsin inhibitors — soybean trypsin inhibitor (SBTI) and benzamidine. The enzyme was active over a broad pH range (from 7.5 to at least pH 10.0) at 25 °C and was stable from pH 4.0 to pH 10.0 when incubated at 20 °C, with a maximum at pH 8.0. The optimum temperature for the hydrolysis of dl-BAPNA by the chinook salmon enzyme was 60 °C, however, the enzyme was unstable at temperatures above 40 °C. The molecular mass of the chinook salmon trypsin was estimated as 28 kDa by SDS–PAGE.     

Major histocompatibility complex differentiation in Sacramento River chinook salmon

The chinook salmon of the Sacramento River, California, have been reduced to a fraction of their former abundance because of human impact and use of the river system. Here we examine the genetic variation at a major histocompatibility complex class II exon in the four Sacramento chinook salmon runs. Examination of the alleles found in these and other chinook salmon revealed nucleotide patterns consistent with selection for amino acid replacement at the putative antigen-binding sites. We found a significant amount of variation in each of the runs, including the federally endangered winter run. All of the samples were in Hardy-Weinberg proportions. A significant amount of genetic differentiation between runs was revealed by several measures of differentiation. Winter run was the most genetically divergent, while the spring, late-fall, and fall runs were less differentiated.     

Phagocytosis of Loma salmonae (Microsporidia) spores in Atlantic salmon (Salmo salar), a resistant host, and chinook salmon (Oncorhynchus tshawytscha), a susceptible host

The in vitro phagocytosis of Loma salmonae spores by macrophages of Atlantic salmon and two strains of chinook salmon were investigated. Opsonisation of L. salmonae with plasma factors increased uptake by head kidney macrophages. Macrophages of Atlantic salmon, which are resistant to the parasite, had a significantly higher phagocytic index (PI) than those of chinook salmon, a susceptible species. This may indicate a possible mechanism contributing to resistance in Atlantic salmon or that L. salmonae is able to evade or suppress initial binding by macrophages of chinook. Non-specific binding or lectinophagocytosis was also suggested by significantly higher PI of spores from EDTA treated plasma when compared with no plasma or heat treated plasma. In comparison, uptake of Baker's yeast Saccharomyces cerevisiae by phagocytes was not significantly different between fish species and strains for all treatments.     

Plasticity of smolting in spring chinook salmon: relation to growth and insulin-like growth factor-I

In two year classes of Willamette River spring chinook salmon, reared at the Willamette Hatchery, and two groups of Yakima River spring chinook salmon, one sampled from the Yakima River and the other reared in a hatchery, fish which had relatively high growth rates in the summer–autumn period smolted in the autumn as measured by increases in gill Na⁺ K⁺ AT Pase activity. In contrast, groups with relatively low growth rate did not smolt in the autumn. Plasma levels of insulin-like growth factor-I (IGF-I) showed discrete differences between groups, with high levels associated with increased gill Na⁺ K⁺ AT Pase activities. These results demonstrate that smolting is plastic in spring chinook salmon, occurring in the autumn or the spring. In addition, smolting appeared to be related to growth rate; however, the relationships shown were correlational and causal mechanisms were not elucidated. Yet, the results do indicate a relationship between growth, an endocrine growth factor and smolting, suggesting a mechanistic link between developmental plasticity and the environment mediated by the endocrine system.