A major effect quantitative trait locus for whirling disease resistance identified in rainbow trout (Oncorhynchus mykiss).

Whirling disease, caused by the pathogen Myxobolus cerebralis, leads to skeletal deformation, neurological impairment and under certain conditions, mortality of juvenile salmonid fishes. The disease has impacted the propagation and survival of many salmonid species over six continents, with particularly negative consequences for rainbow trout. To assess the genetic basis of whirling disease resistance in rainbow trout, genome-wide mapping was initiated using a large outbred F(2) rainbow trout family (n=480) and results were confirmed in three additional outbred F(2) families (n=96 per family). A single quantitative trait locus (QTL) region on chromosome Omy9 was identified in the large mapping family and confirmed in all additional families. This region explains 50-86% of the phenotypic variance across families. Therefore, these data establish that a single QTL region is capable of explaining a large percentage of the phenotypic variance contributing to whirling disease resistance. This is the first genetic region discovered that contributes directly to the whirling disease phenotype and the finding moves the field closer to a mechanistic understanding of resistance to this important disease of salmonid fish.

Euthanization methods influence cytokine mRNA expression levels in age 0 year Oncorhynchus mykiss.

Significant differences in cytokine transcription were found between Oncorhynchus mykiss euthanized using the pharmacological agents MS-222 v. benzocaine and also when contrasting death induced by carbon dioxide asphyxiation v. physical methods (cervical dislocation). This study highlights the need to consider the potentially confounding effect of euthanization method on gene expression data.

Development of PCR assays to detect iridovirus infections among captive and wild populations of Missouri River 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.

PCR assay for improved diagnostics of epitheliotropic disease virus (EEDV) in lake trout Salvelinus namaycush.

Epizootic epitheliotropic disease virus (EEDV) has caused catastrophic losses among hatchery-reared juvenile lake trout Salvelinus namaycush since the early 1980s and remains a major impediment to lake trout restoration in the Great Lakes basin of the USA. Although EEDV has been tentatively designated as a herpesvirus based upon morphological criteria, further characterization of the virus and development of improved detection methods have been hampered by the inability to propagate the virus in cell culture. Recently obtained sequence data for a region of the putative terminase gene from EEDV as well as the related Salmonid herpesvirus 1 and 2 have permitted the development of a polymerase chain reaction (PCR) assay for specific detection of EEDV. The new EEDV PCR demonstrated both an excellent analytic sensitivity and specificity and detected viral DNA as present in the skin of lake trout during periods of active viral outbreaks. In addition, EEDV DNA was detected among healthy appearing juveniles and in the ovarian fluids of spawning adults. Here we describe the development and initial validation steps of the EEDV PCR as a replacement for current diagnostic methods that require virus extraction from the skin, partial purification by isopycnic centrifugation, and visualization of negatively-stained virions by electron microscopy.

Antibody response of two populations of common carp, Cyprinus carpio L., exposed to koi herpesvirus.

Common carp, Cyprinus carpio L., exposed to koi herpesvirus (KHV) may become persistently infected and populations containing such virus-infected individuals may transmit the virus to other fish when co-habited. Detection of virus-infected fish in a population is thus critical to surveillance and control programmes for KHV. A study was therefore designed to detect anti-KHV serum antibodies, with an enzyme-linked immunosorbent assay, in common carp following experimental exposures to KHV under varying environmental conditions. The study determined that a proportion of fish within a population experimentally exposed to KHV (at least 10-25%) develop high antibody titres (1/1600 or greater) to the virus, and this immunological response was detectable for several months (observed at the termination of the experiments at 65, 46 and 27 weeks post-exposure). Furthermore, this response was detected in one population of fish that did not succumb to a high level of mortality when maintained at water temperatures that were non-permissive for KHV. Elevating the water temperatures to permissive conditions for KHV resulted in recurrence of disease despite the presence of anti-virus antibodies, suggesting that serum antibodies alone are not protective under the conditions of our trials.

Essential veterinary education in fish health and disease: a global perspective.

Fish are the largest class of vertebrates, with over 25,000 estimated species and subspecies. Fish have evolved unique anatomical and physiological adaptations, when compared to terrestrial vertebrates, for life in a range of aquatic environments. Interest in aquatic animal health has been recorded in Eastern and Western cultures for more than 2,000 years. In recent times, there has been an increase in the numbers of aquatic animals being used as companion animals or pets, for food and in laboratories, as well as in restoration and conservation programmes. There has also been a corresponding increase in concern for their health and welfare. Moral and ethical considerations require the optimisation of husbandry practices and advances in aquatic animal health for these animals. As with other vertebrates, veterinarians are best equipped to meet the challenges for aquatic animal health from clinical, scientific and legal perspectives. To accomplish this goal, veterinary education must incorporate aquatic animal health throughout graduate curricula, create advanced postgraduate training opportunities, and support a continuum of professional development opportunities for all levels of aquatic animal health expertise.

Evaluation of a range of doses of ultraviolet irradiation to inactivate waterborne actinospore stages of Myxobolus cerebralis.

The ability of a range of doses of ultraviolet irradiation (UV) to inactivate the waterborne actinospore or triactinomyxon stages (TAMs) of Myxobolus cerebralis was evaluated by infectivity for juvenile rainbow trout Oncorhynchus mykiss. TAMs were UV-irradiated using a low pressure mercury vapour lamp collimated beam apparatus. All doses 40, 80, 120 and 160 mJ cm(-2) were found to completely inactivate the TAMs as demonstrated by the absence of microscopic lesions, myxospores and parasite DNA detected by quantitative PCR (qPCR) among rainbow trout 5 mo post-exposure. In contrast, rainbow trout receiving the same concentrations of untreated TAMs (1000 fish(-1)) developed clinical signs of whirling disease at 2 mo post-exposure and had severe microscopic lesions, high myxospore counts and high qPCR values when examined at 5 mo following exposure to the parasite.

Temperature-dependency of Betanodavirus infection in SSN-1 cell line.

This study examined the in vitro effects of temperature on Betanodavirus infection in the SSN-1 cell line. A Betanodavirus isolated from moribund sea bass fry Dicentrarchus labrax farmed in the Adriatic Sea and characterised as a RGNNV (Redspotted Grouper Nervous Necrosis Virus) genotype was used. Virus-infected SSN-1 cells were incubated at temperatures between 10 and 30 degrees C and observed for cytopathic effects daily for 15 d. Cell-free and cell-associated viral growth were evaluated by 50% tissue culture infectious dose (TCID50) titration at 0, 24, 48, 72, 96, 144, 192, 240, 312 and 360 h post-infection. Virus replication was observed at all temperatures from 15 to 30 degrees C. The optimal temperature for virus growth was 25 degrees C. A temperature of 10 degrees C was detrimental to the growth of the SSN-1 cells and cell death interfered with interpretations of viral growth. The isolate of Betanodavirus from Italian sea bass in this study demonstrates a different temperature range for growth compared to previous reports for related Betanodavirus strains, most likely due to an adaptation to the normal environmental temperatures of the host fish species of origin.

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.

Humoral immunoglobulins of the white sturgeon, Acipenser transmontanus: partial characterization of and recognition with monoclonal antibodies.

White sturgeon (Acipenser transmontanus) immunoglobulin (Ig) was purified from serum by two methods, ion-exchange chromatography and gel filtration and precipitation of the euglobulin fraction. The purity of these immunoglobulin preparations was confirmed by gel electrophoresis. Sequence analysis of the N-terminal amino acids confirmed that the purified protein was immunoglobulin. The major portion of the immunoglobulin preparation consisted of two proteins with estimated molecular weights (m.w.) of 870 and 170 kDa. The m.w. of the H- and L-chains of the purified Ig were 73 and 27-30 kDa, respectively, as determined by SDS-PAGE. Ion-exchange purified Ig was used to immunize mice for the production of monoclonal antibodies. This resulted in the production of six stable hybrids that recognized sturgeon Ig, two specific for heavy chain and four specific for light chain. The two anti-H-chain mabs were highly specific for white sturgeon Ig while all four anti-L-chain mabs cross reacted with Ig from green sturgeon (A. medirostris), Atlantic sturgeon (A. oxyrhynchus oxyrhynchus), shovelnose sturgeon (Scaphirhynchus platorynchus), and paddlefish (Polyodon spathula), (all Chondrosteans), but not with channel catfish (Ictalurus punctatus), rainbow trout (Oncorhynchus mykiss) or striped bass (Morone saxatilis). The mabs were used to enumerate the percentage of sIg+ lymphocytes in the peripheral blood of white sturgeon by flow cytometry. The percentage of cells positively stained with the mabs ranged from 12 to 28%. In a comparison of mabs with polyclonal rabbit anti-sturgeon Ig serum by ELISA the mabs produced a larger signal and less background than the polyclonal serum.

Effect of water temperature on the development, release and survival of the triactinomyxon stage of Myxobolus cerebralis in its oligochaete host.

The development of the triactinomyxon stage of Myxobolus cerebralis and release of mature spores from Tubifex tubifex were shown to be temperature dependent. In the present work, the effect of temperature over a range of 5-30 degrees C on the development and release of the triactinomyxon stages of M. cerebralis was studied. Infected T. tubifex stopped releasing triactinomyxon spores 4 days after transfer from 15 degrees C to 25 degrees C or 30 degrees C. Transmission electron microscopic examinations of the tubificids held at 25 degrees C and 30 degrees C for 3 days showed that all developmental stages degenerated and transformed to electron-dense clusters between the gut epithelial cells of T. tubifex. In contrast, tubificid worms held at 5 degrees C and 10 degrees C examined at the same time were heavily infected with many early developmental stages of triactinomyxon. At 15 degrees C, the optimal temperature for development, maturing and mature stages of the parasite were evident. Infected T. tubifex transferred from 15 degrees C to 20 degrees C stopped producing triactinomyxon spores after 15 days. However, 15 days at 20 degrees C was not sufficient to destroy all developmental stages of the parasite. When the tubificid worms were returned to 15 degrees C, the one-cell stages and the binucleate-cell stages resumed normal growth. It was also demonstrated that T. tubifex cured of infection by holding at 30 degrees C for 3 weeks and shifted to 15 degrees C could be re-infected with M. cerebralis spores. The waterborne triactinomyxon spores of M. cerebralis did not appear to be as short-lived as previously reported. More than 60% of experimentally produced waterborne triactinomyxon spores survived and maintained their infectivity for rainbow trout for 15 days at water temperatures up to 15 degrees C. In natural aquatic systems, the triactinomyxon spores may survive and keep their infectivity for periods even longer than 15 days.

Comparison of 18S and ITS-1 rDNA sequences of selected geographic isolates of Myxobolus cerebralis.

Myxobolus cerebralis, the myxosporean parasite-causing salmonid whirling disease, was first reported among rainbow trout (Oncorhynchus mykiss) in Germany in 1903. The parasite was reported for the first time in North America in 1958 among hatchery-reared trout in the eastern USA, presumably arriving with frozen trout shipments from Europe. A comparison of 18S and ITS-1 ribosomal DNA sequences was conducted to identify potential strain differences between selected geographic isolates of this parasite from Europe and North America. Only fourteen of 1700 base pairs were different in the 18S rRNA gene from isolates obtained from California and West Virginia in the USA, and the Federal German Republic. No evidence for strain differences was obtained from ITS-1 sequences that were found to be identical among all parasite isolates. This finding is consistent with the hypothesis that the parasite was recently introduced to the USA from Europe.

Production of viable cultures of Flavobacterium psychrophilum: approach and control.

Although the fish pathogen Flavobacterium psychrophilum is a major source of concern in salmonid hatcheries, few studies have been conducted on its pathogenicity. Difficulties are often experienced when trying to control or quantify standard procedures for in vitro culture of the bacterium. Plate enumeration and counting chamber enumeration combined with epifluorescent microscopy with fluorescent dyes determined that no more than 25% of the bacterial cells present in the cultures were able to produce colonies on agar media. This was strongly dependent upon different medium components. Tryptone-enriched Anacker and Ordal medium proved more suitable than tryptone-yeast extract-salts with skimmed milk. Adding horse serum and trace elements in controlled proportions offered the most reproducible results. Viable but nonculturable forms were apparently not responsible for the difficulties in production of F. psychrophilum, but the cells were highly susceptible to osmotic conditions. Improvements in the media and careful handling of the bacteria in isotonic suspension media resulted in predictable production of viable bacteria and allowed an absorbance/colony-forming-units relation curve to be established.

Rapid serotyping of infectious pancreatic necrosis virus by one-step enzyme-linked immunosorbent assay using monoclonal antibodies.

A one-step ELISA has been developed for detection and serotyping of infectious pancreatic necrosis virus (IPNV) in infected cell cultures using monoclonal antibodies (mAb) raised against strains representing the Sp, Ab and VR 299 serotypes of IPNV. This assay uses a serotype-specific mAb as capture and a mAb directed against a common epitope as detector antibody. Avidin-peroxidase was employed for amplification. The assay was performed in 90 min by simultaneous incubation of the samples, the biotin labelled mAb and the avidin-peroxidase, and detected 37 ng/ml of purified virus. Serotyping of 34 isolates from different areas of Europe, Asia and America showed a total concordance with the results obtained by the neutralization assay.

Effect of the microsporidian Enterocytozoon salmonis on the immune response of chinook salmon.

The protozoan parasite Enterocytozoon salmonis is an intranuclear microsporidian of salmonid mononuclear leukocytes. Experimental infections were initiated in chinook salmon to determine the effects of parasitism on selected host immune functions. The humoral antibody response to dinitrophenylated-keyhole limpet hemocyanin and the in vitro blastogenic responses of isolated mononuclear leukocytes to mitogens (concanavalin A, lipopolysaccharide and phytohemagglutinin-P) were evaluated. The humoral response as detected by enzyme-linked immunosorbent assays was suppressed following infection. The degree of suppression increased as the severity of the infection progressed. Additionally, the response to mitogen-induced lymphoproliferation was impaired. These results suggest that infection with E. salmonis may cause suppression of host cell immune functions, thus increasing the susceptibly of infected fish to other pathogens.

Molecular detection of an Ehrlichia-like agent in rainbow trout (Oncorhynchus mykiss) from Northern California.

Ehrlichia DNA was identified by nested PCR in rainbow trout (Oncorhynchus mykiss) collected from a creek in northern California where Potomac horse fever is endemic. Ehrlichia DNA was found in tissues from several organs including the gills, heart, spleen, liver, kidneys and intestine of trout and from three different adult digenetic trematodes (Deropegus sp., Crepidostomum sp., Creptotrema sp.) parasitizing the gallbladder and/or the intestine of the trout. Sequencing of PCR-amplified DNA from the 16S rRNA gene indicated that the source organism was most closely related to the sequences of E. risticii (level of sequence similarity 96.0%), the SF agent (95.9%), E. sennetsu (95.8%), and Neorickettsia helminthoeca (95.3%). The data suggest that trout and parasitic trematodes may be involved in the epidemiology of an Ehrlichia-like agent belonging to the E. sennetsu genogroup. Whether the fish agent infects horses, dogs, or human beings, and whether it causes disease, remain to be determined.

The role of epidemiology in the prevention, diagnosis, and control of infectious diseases of fish.

Epidemiologic methods are essential to understanding infectious diseases in aquaculture. Unfortunately, many of these methods are poorly understood or not utilized by fish-health scientists and aquaculturists -- often because of the lack of contact with epidemiologists who are willing to investigate fish diseases. In this paper, we describe direct interactions between epidemiologists and fish-health specialists that have resulted in an improved understanding of the causes and management of infectious diseases in aquaculture. We focus on risk-factor studies, risk analysis and infectious-disease modeling, evaluation of diagnostic tests and experimental studies. We also describe characteristics of confined fish populations that make them ideal for developing and testing epidemiologic models and the theoretical and practical challenges of designing and conducting epidemiologic studies in fish farms. Throughout our presentation, emphasis is given to characteristics, opportunities and problems associated mainly with conducting epidemiologic studies to intensive aquaculture systems. We conclude that the development of increased cooperation among epidemiologists, fish-health scientists and aquaculturists will be mutually beneficial and, therefore, efforts for such collaboration should be initiated from all parties involved.

Mortality and recovery of runt white sturgeon (Acipenser transmontanus) in a commercial farm in California, USA.

We investigated the effect of raising runt white sturgeon (Acipenser transmontanus) separately from dominant fish during the initial stages of grow-out in a commercial farm. Runt fish are poor-growers, have underdeveloped muscle mass, swim slowly and are more-frequently found at the top of the water column. The objective of the study was to describe the mortality and recovery rates (and their determinants) of white-sturgeon runts after separating them from dominant fish. Runt white sturgeon were stocked into twelve 2 m x 2 m rectangular tanks and graded periodically during a follow-up of 46-102 days. Overall mortality rates ranged from 0.3 to 7 dead fish per 1000 sturgeon-days at risk and overall recovery rates from 3.9 to 13.5 recovered fish per 1000 sturgeon-days at risk. Period-specific mortality and recovery rates increased over time. The period-specific mortality rates for all three periods were significantly higher for tanks of runts originating from grow-out tanks with high mortality (p-values: first period = 0.06; second period = 0.09; third period = 0.03), but were similar for tanks of runts of high- and low-mean initial weight. The period-specific recovery rates were significantly higher in runts originating from high-mortality grow-out tanks only for the third period (p = 0.05) but not the first and second periods (p-values = 0.33 and 0.25, respectively). Recovery rates were significantly higher in the higher-mean-weight runts tanks for the first and third period but not for the second (p-values: first period = 0.02; second period = 0.65; third period = 0.06). We concluded that the proportion of runts that recover during a 46-89 day period is substantial (16-58%); therefore, it might be worthwhile growing such fish separately in a fish farm for about three months. Financial analysis showed that this practice was profitable, if the value of white sturgeon fish for the farm exceeded $2.05 per kg.

Growth of white sturgeon (Acipenser transmontanus) following recovery from the stunted stage in a commercial farm in California, USA.

Runt white sturgeon (Acipenser transmontanus) develop during grow-out and are characterized by atrophied muscles and decreased growth. Our first objective was to compare the growth (and body condition) of previously-runt white sturgeon after they recovered from the runt state and sturgeon that had never been runts. On 12 occasions, recovered runts and age- and size-matched controls that had never been runts were tagged and put in a tank that already contained fish of similar age and size. Tagged groups were followed for 119-134 days. Median relative growth rates (RGRs) of the recovered runts were significantly (p < or = 0.05) higher than those of the controls in three tanks. Multiple linear regression was used to model final weight as a function of initial weight and status (recovered runt or control). Status was not significantly related (p = 0.71) to final weight, after adjusting for initial weight, "tank" and time of follow-up. Our second objective was to determine factors that influenced the loss of tags by white sturgeon during the follow-up period. Logistic regression analysis indicated that higher initial weight and being a control fish might have been associated with losing both tags. We concluded that once white sturgeon runts recovered and started growing, they grew at least as well as fish that had never been runts.

A nested polymerase chain reaction for the detection of genomic DNA of Myxobolus cerebralis in rainbow trout Oncorhynchus mykiss.

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.