Conservation of native species is challenged by the introduction of non-native pathogens and diseases into aquatic and terrestrial environments worldwide. In the Yellowstone Lake basin, Yellowstone National Park, the invasive parasite causing salmonid whirling disease Myxobolus cerebralis (Hofer) has been identified as one factor contributing to population declines of native Yellowstone cutthroat trout Oncorhynchus clarkii bouvieri (Jordan & Gilbert). In 2002 and 2003, we examined relationships between the stream environment and severity of M. cerebralis infection in native trout. Coefficients of variation of environmental features were calculated to examine variability. Ten years later, we reassessed infection levels at 22 tributaries broadly across the system. Results of principal component analysis (PCA) of physical features (2003) were negatively correlated with infection severity, mostly in lower jaw cartilage of cutthroat trout, and PCA of chemical features (and temperature) correlated with infection severity in cranial cartilage. Pelican Creek, where M. cerebralis prevalence and severity was high 2002-2003, remained high in 2012. We did not find evidence that the parasite had dispersed further within the system. Variable environmental features (physiological stress) across short spatiotemporal scales within a stream or season may possibly predispose salmonids to infection in the wild and facilitate parasite establishment.

Iridovirus infections of the integument were associated with disease and mortality among hatchery-reared populations of juvenile pallid sturgeon Scaphirhynchus albus and shovelnose sturgeon S. platorynchus from the Missouri River. Virus-infected cells in the integument of fins and body were greatly enlarged, possessed pleomorphic and eccentric nuclei, and exhibited an amphophilic to eosinophilic staining of the cytoplasm in hematoxylin-and-eosin-stained sections. Virus particles found in the host cell cytoplasm were composed of an outer hexagonal capsid measuring 254 nm in diameter and surrounding a dense nucleoid. Despite numerous attempts, the virus could not be propagated on routine cell lines used in fish viral diagnostics or from established cell lines from white sturgeon Acipenser transmontanus, pallid sturgeon, or shovelnose sturgeon. Bath exposures of healthy juvenile pallid sturgeon to a crude extract or a 0.45-microm-filtered extract from the fins of infected fish resulted in transmission of the virus and mortality. At water temperatures of 15 degrees C, the first deaths occurred at approximately 1 month; mortality peaked between 50 and 60 d postexposure, after which surviving fish recovered. Presence of the virus was confirmed among dead and moribund pallid sturgeon by both histology and detection of viral DNA by polymerase chain reaction methods. Feeding of infected tissues and cohabitation with virus-infected shovelnose sturgeon also resulted in successful virus transmission to juvenile pallid sturgeon. Virus infections among experimentally exposed pallid sturgeon that recovered from clinical episodes persisted for at least 8.5 months, and these apparently healthy fish transmitted the virus and disease to juvenile pallid sturgeon by cohabitation. The newly described Missouri River sturgeon iridovirus (MRSIV) as found in pallid sturgeon and shovelnose sturgeon shares many properties with a group of iridoviruses associated with serious skin and gill infections in several species of sturgeon.

The Missouri River sturgeon iridovirus (MRSIV) is an important factor contributing to losses during the hatchery rearing of juvenile pallid Scaphirhynchus albus and shovelnose S. platorynchus sturgeon. As the virus has not been isolated in cell culture, current detection procedures rely upon a combination of light and electron microscopy. Detection of characteristic virus-infected cells in the integument, usually of the fins, in hematoxylin and eosin (H&E)-stained tissue sections provides a presumptive finding. Confirmation requires observation by electron microscopy of characteristic doubly enveloped hexagonal virions of the appropriate size in the host cell cytoplasm. To improve these diagnostic procedures, a conventional polymerase chain reduction (PCR) assay was developed as a sensitive and specific method for detection of MRSIV DNA as found in numerous tissues of both naturally and experimentally infected pallid and shovelnose sturgeon. Sequences of amplicons obtained from testing of wild-caught shovelnose sturgeon and juvenile pallid sturgeon during hatchery outbreaks were identical, suggesting that the viruses found in both sturgeon are similar or closely related. In addition, a TaqMan PCR was developed that allowed estimates of the concentrations of MRSIV DNA present in the tissues of pallid and shovelnose sturgeon during acute and persistent infection. These new PCR assays are improved methods to detect MRSIV, but equally importantly, they provide insights into to the biology of the agent for more effective management of viral diseases in captive and wild Missouri River sturgeon populations.

The impacts of whirling disease on the aquaculture of trout over the past 80 years has resulted in the development of simple to more complex methods to detect the causative agent Myxobolus cerebralis. With the absence of effective treatments, efficient detection of this pathogen is critical. It is our goal to give a historical perspective to these developments while discussing the relative merits of the different techniques.

Whirling disease is a chronic inflammatory disease in salmonid fish caused by the myxosporean parasite Myxobolus cerebralis. The disease, first recognized in cultured rainbow trout Oncorhynchus mykiss, is characterized by the radical tail-chasing behavior of infected fish. The parasite is enzootic in free ranging and cultured populations of trout in areas of salmonid aquaculture, except South America. Recent reports of severe population declines among wild trout associated with whirling disease has caused great concern and renewed interest in this pathogen. The parasite has a sequential affinity for the skin, nerves, and, finally, skeletal cartilage, in the fish host. Abundant cartilage in the skeleton of young trout renders them extremely susceptible to the effects of the disease. Most salmonid species are susceptible to infection with M. cerebralis, but susceptibility varies among species and may also vary among strains and individual fish within a population or a similarly exposed group. Rainbow trout and anadromous steelhead are highly susceptible to whirling disease. The severity of the disease and mortality are related to age of the fish, when first exposed, and parasite dose. Trophozoite stages of the parasite lyse cartilage, cause an inflammatory response, and may interfere with normal bone deposition in the salmonid host. In severely infected fish, growth rates are depressed during active infection, and behavioral effects and severe skeletal deformities compromise functions such as swimming and feeding. The effects of M. cerebralis on the salmonid host are determined by factors such as species, age, size, parasite dose, immune response, and water temperature. Understanding the interaction of these factors in waters where M. cerebralis is present is critical to the future management of healthy wild trout populations.

Two strains of rainbow trout Oncorhynchus mykiss and one strain of steelhead (anadromous rainbow trout) previously shown to demonstrate resistance to the myxosporean pathogen Ceratomyxa shasta were found to be susceptible to experimentally induced infections with the myxosporean Myxobolus cerebralis. Laboratory exposures to waterborne infectious stages (triactinomyxons) of M. cerebralis of both strains of rainbow trout and the steelhead resulted in clinical signs of whirling disease, including the characteristic tail chasing and black tail, approximately 42-49 d after exposure at a water temperature of 15 degreesC. At 5 months postexposure, the severity of microscopic lesions and spore concentrations in the head cartilage of all salmonids resistant to C. shasta were evaluated. The Iron Gate steelhead and the Pit River rainbow trout that were resistant to C. shasta had lesion scores and spore counts similar to those of control rainbow trout known to be susceptible to C. shasta. Evidence of some resistance to M. cerebralis among the Deschutes River strain of rainbow trout was demonstrated by lower lesion scores and mean spore counts than were seen in the control rainbow trout strain. In general, however, the mechanisms of resistance that have developed among certain salmonid populations to one myxosporean, C. shasta, do not extend to protection or resistance to a second myxosporean, the causative agent of salmonid whirling disease.

The susceptibility of three species of anadromous salmonids to whirling disease was examined after their experimental exposures to the infectious stages of Myxobulus cerebralis. Chinook salmon Oncorhynchus tshawytscha exposed as alevins were very susceptible to infection; the appearance of clinical signs, prevalence of infection, severity of microscopic lesions, and spore counts at 130 d postexposure are similar to those of age-matched rainbow trout O. mykiss exposed at the same dose. In contrast, coho salmon O. kisutch demonstrated no clinical signs of infection and had a lower prevalence of infection and spore numbers than did the exposed rainbow trout. A comparison of two strains of steelhead (anadromous rainbow trout), one from an enzootic site (San Lorenzo River) where M. cerebralis has been present for the past 35 years and a second from a site where the parasite is not found (Dry Creek), showed them both to be highly susceptible to experimental infections with M cerebralis. These controlled experimental exposures demonstrated that although certain species of anadromous salmonids (e.g., coho salmon) may resist the ill effects of whirling disease, other species (chinook salmon and steelhead) are highly susceptible to infection and disease. We anticipate that severe and negative impacts at the population level might occur among certain anadromous salmonids when conditions favor exposure of early life stages to high levels of M cerebralis.

The susceptibility of rainbow trout Oncorhynchus mykiss and brown trout Salmo trutta to Myxobolus cerebralis, the cause of salmonid whirling disease, was assessed following dosed exposures to the infectious stages (triactinomyxons). Parallel groups of age-matched brown trout and rainbow trout were exposed to 10, 100, 1000 or 10,000 triactinomyxons per fish for 2 h and then placed in aquaria receiving single pass 15 degrees C well water. Severity of infection was evaluated by presence of clinical signs (whirling and/or black tail), prevalence of infection, severity of microscopic lesions, and spore counts 5 mo after exposure. Clinical signs of whirling disease, including a darkened caudal region (black tail) and radical tail chasing swimming (whirling), occurred first among rainbow trout at the highest dose at 6 to 7 wk post exposure. Black tail and whirling occurred among rainbow trout receiving 1000 and 100 triactinomyxons per fish at 8 to 9 wk post exposure. Only 1 of 20 fish had a black tail among rainbow trout receiving 10 triactinomyxons per fish, although 30% of the fish were infected at 5 mo post exposure. Black tails were observed in brown trout at 1000 and 10,000 triactinomyxons per fish beginning at 11 and 7 wk post exposure, respectively. There was no evidence of the tail chasing swimming (whirling) in any group of brown trout. The prevalence of infection, spore numbers, and severity of microscopic lesions due to M. cerebralis among brown trout were less at each exposure dose when compared to rainbow trout. Infections were found among rainbow trout at all doses of exposure but only among brown trout exposed to doses of 100 triactinomyxons per fish or greater. Risk of infection analyses showed that rainbow trout were more apt to be infected at each exposure dose than brown trout. Spore counts reached 1.7 x 10(6) per head among rainbow trout at the highest dose of exposure compared to 1.7 x 10(4) at the same exposure dose among brown trout. Spore numbers increased with dose of exposure in rainbow trout but not in brown trout. As microscopic lesion scores increased from mild to moderate, spore numbers increased in rainbow trout but not brown trout. The mechanisms by which brown trout resist infections with M. cerebralis were not determined. Cellular immune functions, including those of eosinophilic granular leukocytes that were more prominent in brown trout than rainbow trout, may be involved.

Laboratory exposures to the infectious stages (triactinomyxons) of Myxobolus cerebralis demonstrated a range of susceptibility to whirling disease among four species of inland salmonids. Replicate groups of each species were exposed to two concentrations of triactinomyxons, a low dose (100-200 per fish) and a high dose (1,000-2,000 per fish). Exposed fish were evaluated for clinical signs, for severity of microscopic lesions at 35 d, 2 and 5 months, and for spore concentrations in the head cartilage at 5 months. A standard strain of rainbow trout Oncorhynchus mykiss matched for age served as a susceptible species control. Rainbow trout, westslope cutthroat trout O. clarki lewisi, Yellowstone cutthroat trout O. clarki bouvieri, and bull trout Salvelinus confluentus were susceptible to M. cerebralis infections. Clinical signs, including radical swimming ("whirling") and black tails, were observed at 7 weeks postexposure among rainbow and cutthroat trout challenged at 3 weeks of age. Clinical signs were rare among bull trout exposed at an age of 4 weeks and absent among rainbow and cutthroat trout exposed at 3 months posthatch. Most rainbow, cutthroat, and bull trout were found to be infected when examined at 5 months postexposure. The most severe microscopic lesions among infected fish at 5 months postexposure were found among rainbow trout. Cutthroat trout had less severe lesions, bull trout had mild infections, and no evidence of infection was found among Arctic grayling Thymallus arcticus. Mean spore concentrations among infected fish correlated with the severity of microscopic lesion scores. Rainbow trout had mean concentrations of spores in head cartilage reaching 10(6), whereas more resistant species such as bull trout had 10(4) spores; no spores were found among Arctic grayling at 5 months postexposure.

Summary: Whirling disease of rainbow trout is caused by Myxobolus cerebralis, a myxozoan parasite possessing a life cycle well adapted to the natural environments where salmonid fish are found. Whirling disease was first described in Europe in 1898 among farmed rainbow trout but recent occurrences have been devastating to wild trout in North America. The disease is considered a major threat to survival of wild rainbow trout in the intermountain west of the United States. Difficulties in containing the spread and potentially eliminating the pathogen are tied to features of a complex life cycle involving two hosts, the salmonid fish and an aquatic oligochaete. Details of the morphologic development of the parasite have been described in each host but only now are we beginning to appreciate the breadth of interactions between these developmental forms and the sequential responses of the host. Fundamental mechanisms of the recognition and attachment of the parasite to the hosts, how host immunity is evaded and the unknown influences of environmental factors all contribute to a rather poor understanding of the biology of the parasite. Although the biology and ecology of the salmonid host are better known than for the oligochaete host, our knowledge is inadequate to interpret their complex interactions with the parasite. This uncertainty precludes the development of effective management activities designed to enhance the viability and productivity of wild trout populations in M. cerebralis‐ positive river systems. Improving our understanding of the hosts, the parasite and the environmental factors determining their interaction should provide for more focused and effective control methods for containing the spread and devastating effects whirling disease is causing to our wild trout populations.

A nested polymerase chain reaction (PCR) test was developed to amplify a segment of the 18S rRNA gene from Myxobolus cerebralis, the agent causing whirling disease in salmonid fish. The PCR amplifies a 415 bp amplicon that was identified by dideoxynucleotide terminated sequencing to be identical to the known 18S rDNA sequence of M. cerebralis. There was no amplification of genomic DNA from 4 other myxosporean parasites of salmonid fish from the genus Myxobolus including M. arcticus, M. insidiosus, M. neurobius, and M. squamalis. The efficacy of the PCR test to detect early infections was demonstrated by amplification of the 415 bp fragment from experimentally exposed rainbow trout Oncorhynchus mykiss at 2 h and at 1, 2, and 3 wk postexposure to actinosporean stages (triactinomyxons) of M. cerebralis. In contrast, standard microscopic examinations of stained tissue sections of the same fish used for PCR were less reliable in detecting the presence of the parasite. Additional examinations of fish 5 mo postexposure, after sporogenesis had occurred, found the PCR to be a more reliable indicator of infection than pepsin-trypsin digest (PTD) method, particularly when trout were experimentally exposed to low levels of the infectious stages of the parasite. The PCR was able to amplify to detectable levels the equivalent of a single sporoplasm of M. cerebralis as found in a tissue sample. This test improves the detection of M. cerebralis because it can detect the presence of the parasite: (1) in both hosts, (2) in all known stages of its life cycle, and (3) at lower thresholds than currently used diagnostic methods. Lastly, the PCR test is less susceptible to morphological misidentifications of the spores that can occur with current microscopic procedures.

Proliferative kidney disease (PKD) is one of the most economically important diseases among commercially reared rainbow trout (Oncorhynchus mykiss) in Europe and causes significant losses among Pacific salmon and rainbow trout populations in western North America. The parasite that causes PKD is a poorly understood myxosporean (PKX), presumed but not yet proven to be a member of the family Sphaerosporidae, genus Sphaerospora. The disease occurs in both cultured and feral populations of salmonids that come into contact with a 20–25 μm waterborne infective stage. The disease is often seasonally dependent occurring at water temperatures above 15 °C in the summer and fall months of the year. At permissive water temperatures, the first recognizable vegetative or extrasporogonic stages appear about 10–14 days after exposure to the infective stage. They are initially prominent in the blood sinuses of the kidney, are accompanied by a mild hyperplasia of the interstitial hematopoietic cell populations, and multiply via binary fission, endogeny, and plasmotomy, provoking a strong inflammatory response. This diffuse granulomatous response occurs principally in the kidney but is also seen in the spleen and other organs. The major host cell type involved in the lesion is the macrophage, but lymphocytes are also abundant in close proximity to PKX. As the inflammation progresses (6–8 weeks postexposure to the infective stage), the gross renal swelling becomes evident as do other clinical signs including anemia. Mortality in uncomplicated cases of PKD is generally 20% or less but often secondary pathogens or unfavorable environmental conditions coincide with peak periods of PKD and mortalities can reach 95–100%. Compromised renal functions such as macromolecule adsorption and divalent cation excretion are also impaired and this may contribute to the mortality observed among fish with PKD. Generally, by 12–20 weeks postexposure, fish begin, or are in the process of, recovery and the renal hematopoietic and excretory functions return to normal. In fish that have experienced a full clinical episode of the disease, a strong acquired immunity develops. The basis of the immunity is unknown but circulating antiparasite antibodies can be detected as early as six weeks postexposure to the infective stage. A strong cellular component to the immunity is also suspected. No vaccines have yet been developed to control PKD, and only recently have experimental therapies been applied. Both fumagillin DCH, an antibiotic effective against certain microsporidia and myxosporidia, and the arylmethane dye malachite green, have shown some promise as treatments for PKD. Unfortunately, neither drug is licensed for use in the U.S. and both treatments suffer from potential difficulties with drug toxicity, tissue residues, or durg discharges in hatchery effluent waters.

Yearling chinook salmon Oncorhynchus tshawytscha were found to be infected with an intranuclear microsporidium.The primary pathologic response in infected salmon was a marked lymphoblastosis.Although clinical signs and gross pathology were similar to other salmonid fish health problems, the induced cellular changes and prominent nuclear inclusions permitted accurate differentiation and diagnosis.This is the first reported occurrence of this infection in freshwater

Lake trout (Salvelinus namaycush) were reared in water reused through a series of seven tanks for a period of 150 d. Fish loading per tank was based on a flow index of 0.71, giving a cumulative index of 4.97 after the seventh water use. Dissolved oxygen and total ammonia were measured as indicators of water quality. Average dissolved oxygen concentration decreased from 7.6 ppm after the first water use to 3.1 ppm after the seventh use, and average ammonia concentration increased from 0.09 ppm after the first use to 0.94 ppm after the seventh use. Lake trout growth rate began to decline at the fifth water use when dissolved oxygen averaged 3.5 ppm and ammonia 0.75 ppm.