Difficulties in experimental infection studies with Flavobacterium psychrophilum in rainbow trout (Oncorhynchus mykiss) using immersion, oral and anal challenges

This study was carried out in order to try to establish an efficacious and reliable experimental infection model for Flavobacterium psychrophilum, the causative agent of rainbow trout fry syndrome, using contact, oral and anal challenges. Ten F psychrophilum strains of different origin were included. The influence of water temperature, scarification, water quality, stress and growth conditions of the pathogen on the experimental infection was assessed. For each challenge protocol, all strains failed to reproduce disease signs or mortality in rainbow trout (Oncorhynchus mykiss L.) fry. Histological and bacteriological examination of the skin, gills and internal organs of the fish 3 weeks following inoculation were found to be negative. Different hypotheses to explain the inability of the challenge models to reproduce the disease experimentally are discussed.     

Concomitant exposure of rainbow trout fry to Gyrodactylus derjavini and Flavobacterium psychrophilum: effects on infection and mortality of host

Although rainbow trout fry mortality syndrome caused by the bacterium Flavobacterium psychrophilum is widespread in fish farms it is difficult to reproduce infection of rainbow trout in the laboratory using immersion exposure with bacterial suspensions. It has therefore been speculated that ectoparasites could act as enhancers of bacterial infections under natural conditions. In the present study rainbow trout fry were exposed to infections with F. psychrophilum (immersion for 30 min or 10 h) alone, exposed to the ectoparasitic monogenean Gyrodactylus derjavini alone or exposed to both pathogens in combination. Infection levels and host mortality were subsequently monitored to elucidate if the ectoparasitic monogeneans could enhance infection of fish with the bacterium. Immersion of fish in bacterial suspensions alone did not result in infection. Only one fish became infected with the bacterium and this fish belonged to the combination exposure group. The parasite populations increased differently in the various groups and it was found that host mortality was correlated to gyrodactylid infection levels (r=0.94) but not to bacterial exposure. The results emphasise the pathogenicity of the parasite G. derjavini, the relative resistance of intact fish to direct exposure to F. psychrophilum but provide only a weak indication of a possible enhancement of bacterial invasion due to ectoparasitic infections. It cannot be excluded that higher parasite burdens and/or prolonged immersion (more than 10 h) in bacterial suspensions may result in bacterial invasion.     

Histopathological and electron microscopical observations on rainbow trout fry syndrome

Rainbow trout (Oncorhynchus mykiss) fry and fingerlings with clinical signs of rainbow trout fry syndrome, and rainbow trout (0.5 to 3.5 g) with experimentally induced infections with Flavobacterium psychrophilum, were examined histopathologically and electron microscopically. Severe hypertrophy of the spleen and cellular degeneration were consistently observed. Distinctive features of the disease were the loss of definition of the splenic border and its replacement by a loosely structured eosinophilic layer, fibrinous inflammation and intercellular oedema within the spleen, and the presence of numerous filamentous bacteria interspersed throughout the organ.     

In vitro and in vivo detection of infectious pancreatic necrosis virus in fish by enzyme-linked immunosorbent assay

A double antibody enzyme-linked immunosorbent assay (ELISA) was used to detect infectious pancreatic necrosis virus (IPNV). The ELISA detected VR299 strain of IPNV at a dose of 10 to 20 ng of purified IPNV protein or 10(4) TCID50 in tissue culture fluid. Specificity of ELISA was demonstrated by an ELISA inhibition test. The ELISA did not detect infectious hematopoietic necrosis virus. Normal cell culture fluid and virus-non-inoculated rainbow trout (Salmo gairdneri Richardson) homogenate did not react in the test system. The IPNV was detected in rainbow trout fry inoculated with IPNV. Although infective virus titer in fish decreased rapidly 1 week after inoculation, IPNV antigen was detected by ELISA for 15 days. The IPNV antigen was detected in the fish tissue after inactivation of infective virus. The ELISA is a rapid and reliable method for the diagnosis of IPNV infection.     

Comparative susceptibility of Atlantic salmon and rainbow trout to Yersinia ruckeri: Relationship to O antigen serotype and resistance to serum killing

A study was undertaken to compare the virulence and serum killing resistance properties of Atlantic salmon and rainbow trout Yersinia ruckeri isolates. Five isolates, covering heat-stable O-antigen O1, O2 and O5 serotypes, were tested for virulence towards fry and juveniles of both species by experimental bath challenge. The sensitivity of 15 diverse isolates to non-immune salmon and rainbow trout serum was also examined. All five isolates caused significant mortality in salmon fry. Serotype O1 isolate 06059 caused the highest mortality in salmon (74% and 70% in fry and juveniles, respectively). Isolate 06041, a typical ERM-causing serotype O1 UK rainbow trout strain, caused mortalities in both rainbow trout and salmon. None of the salmon isolates caused any mortalities in 150–250 g rainbow trout, and only serotype O2 isolate 06060 caused any significant mortality (10%) in rainbow trout fry. Disease progression and severity was affected by water temperature. Mortality in salmon caused by the isolates 06059 and 05094 was much higher at 16 °C (74% and 33%, respectively) than at 12 °C (30 and 4% respectively). Virulent rainbow trout isolates were generally resistant to sera from both species, whereas salmon isolates varied in their serum sensitivity. Convalescent serum from salmon and rainbow trout that had been infected by serotype O1 isolates mediated effective classical pathway complement killing of serotype O1 and O5 isolates that were resistant to normal sera. Overall, strains recovered from infected salmon possess a wider range of phenotypic properties (relative virulence, O serotype and possession of serum-resistance factors), compared to ERM-causing rainbow trout isolates.     

Effects of Salinity on Yersinia ruckeri Infection of Rainbow Trout and Brown Trout

Abstract

Juvenile rainbow trout Oncorhynchus mykiss and brown trout Salmo trutta acclimated to freshwater or salinities of 9.0‰ or less were exposed to Yersinia ruckeri, the bacterial pathogen that causes enteric redmouth disease (ERM). Both species of fish were kept in the same recirculating systems after bacterial exposure. Rainbow trout mortality was significantly (P < 0.05) different in each salinity: 96.5% in freshwater, 89.5% in water of 1.1‰ salinity, 81.3% in 3.0‰ salinity, and 75.0% in 9.0‰ salinity (model SE = 1.0). All deaths occurred between 3 and 12 d after exposure to Y. ruckeri. Only 2.3% of brown trout in all salinities died, and differences among treatments were not significant. For both fish species, Y. ruckeri was isolated from liver, spleen, and trunk kidney of fish dying during this experiment, and lesions of rainbow trout were consistent with ERM. Yersinia ruckeri was not isolated from brown trout surviving for 21 d after bacterial exposure but was isolated from 3 of 24 surviving rainbow trout; a polymerase chain reaction assay detected the DNA of Y. ruckeri in 3 additional rainbow trout survivors. Neither the lesions of fish with ERM nor the percentage of surviving fish subclinically infected with Y. ruckeri was affected by salinity. Bacterial growth in vitro was not affected by low (≤9.0‰) salinity; however, bacterial adhesion to polystyrene was significantly reduced as salinity increased. Although mortality caused by Y. ruckeri was significantly lower for rainbow trout in water with slightly increased salinity, none of the salinities tested was effective in preventing serious losses caused by this pathogen in recirculating systems.     

Vitamin E-responsive myopathy in rainbow trout fry (Oncorhynchus mykiss).

Unacceptably high mortalities in rainbow trout fry (Oncorhynchus mykiss) six to 10 weeks after they started to feed were recorded in two spring water trout hatcheries in Northern Ireland in May 1989. Muscle degeneration and necrosis were consistent with histopathological findings in both outbreaks, and this myopathy was similar to that previously described in salmonids and other species associated with vitamin E and selenium deficiency. A feed trial was designed to investigate the hypothesis that the vitamin E requirement of rainbow trout fry on these farms was higher than the current minimum recommendations. Three groups of fry were fed diets containing 147, 239 and 532 iu/kg alpha-tocopherol. The mortality in the groups was inversely related to the dietary alpha-tocopherol concentration, and there was severe myopathy in fry fed the diet containing 147 iu/kg alpha-tocopherol, mild myopathy in fry fed 239 iu/kg alpha-tocopherol but no myopathy in fry fed 532 iu/kg alpha-tocopherol.     

In Vitro Infection of Salmonid Epidermal Tissues by Infectious Hematopoietic Necrosis Virus and Viral Hemorrhagic Septicemia Virus

Abstract

The ability of two rhabdoviruses, infectious hematopoietic necrosis virus (IHNV) and viral hemorrhagic septicemia virus (VHSV), to infect fish skin was investigated by in vitro infection of excised tissues. Virus replication was determined by plaque assay of homogenized tissue extracts, and the virus antigen was detected by immunohistology of tissue sections. Gill, fin, and ventral abdominal skin tissues of rainbow trout Oncorhynchus mykiss that had been infected in vitro with a virulent strain of IHNV (193–110) produced substantial increases in virus titer within 24 h. Titers continued to increase up until day 3 of incubation; by this time, virus had increased 1,000-fold or more. This increase in IHNV titer occurred in epidermal tissues of fingerlings and of older fish. In another experiment, IHNV replicated in excised rainbow trout tissues whether the fish had been subject to prior infection with a virulent strain of IHNV (Western Regional Aquaculture Consortium isolate) or whether the fish had been infected previously with an attenuated strain of the virus (Nan Scott Lake, with 100 passes in culture). A virulent strain of VHSV (23/75) replicated effectively in excised gill tissues and epidermal tissues of rainbow trout and chinook salmon O. tshawytscha; however, the avirulent North American strain of VHSV (Makah) replicated poorly or not at all.     

Early Kinetics of Infectious Hematopoietic Necrosis Virus (IHNV) Infection in Rainbow Trout

Abstract

A series of experiments was carried out with infectious hematopoietic necrosis virus (IHNV; 193-110 isolate) in rainbow trout Oncorhynchus mykiss (weight, ～1.2 g) to determine the duration of the patent period and the timing of onset of the infectious periods. We first attempted to transmit IHNV to recipient fish from infected rainbow trout 2–3 d after they had been exposed. No infection transfer occurred despite high titers (10^4.79 to 10^4.91 plaque-forming units 5–8 d postexposure (dpe). To determine the number of secondary cases produced by one infectious individual, we exposed approximately 50 rainbow trout (weight, ～1.5 g) in each of seven replicate tanks to a donor fish that had been infected with virus by bath exposure 3 d earlier. The prevalence of infection in recipient fish rose from 0.84% at 2 dpe to 7.9% at 6 dpe. Maximum incidence (22 cases) occurred between 2 and 4 dpe. No disease-specific mortalities occurred in recipient fish during the experiment. The titer of virus in both recipient and donor fish increased from 2 to 4 dpe. There was a positive correlation between the level of infection among donors and prevalence values among recipient fish (r ² = 0.60). The level of challenge by one infectious fish under the conditions provided was enough for infection transfer from sick cohabitant to susceptible fish but was not enough for initiation of a full-scale epizootic among recipients.     

Relation of Water Temperature to Bacterial Cold-Water Disease in Coho Salmon,Chinook Salmon,and Rainbow Trout

Abstract

The effect of water temperature on the progress of experimentally induced Cytophaga psychrophila infection was investigated in juveniles of coho salmon Oncorhynchus kisutch, chinook salmon O. tshawytscha, and rainbow trout O. mykiss (formerly Salmo gairdneri). A virulent strain of C. psychrophila was administered to fish by subcutaneous injection. Infected fish were held in tanks containing pathogen-free well water at temperatures ranging from 3 to 23°C. Mean times from infection to death of the fish were shortest at 12–15°C, which were the temperatures associated with the shortest time for doubling the population of this bacterium in vitro. Juvenile steelhead (anadromous rainbow trout) injected with viable C. psychrophila cells and held in 22°C water did not become diseased.     

Occurrence of enteric redmouth disease in rainbow trout (Oncorhynchus mykiss) on farms in Croatia

During the spring of 1996 and autumn of 1997 unusual mortality outbreaks among rainbow trout fry and yearlings occurred at two different trout farms, resulting in mortality of 20 and 10 per cent, respectively. Generally, the affected fish, swimming at the water surface, were reluctant to eat and were dark pigmented with visible haemorrhages around and within the oral cavity. Bacterial isolates from moribund fish from both cases were identified as Yersinia ruckeri by standard biochemical tests and API 20E. The isolated strains were found to be sensitive to tetracycline, chloramphenicol, co-trimoxazole, nalidixic acid, flumequine, enrofloxacin, carbenicillin and gentamicin. Microplate agglutination assay confirmed that both isolates belonged to serotype O1. The pathogenicity of the isolated bacteria was confirmed by challenge experiment. Titres of specific antibodies were determined in the sera of survivors. The titre was highest on the 21st day postchallenge and was detectable until the 81st day.     

Multiplication of Infectious Hematopoietic Necrosis Virus in Rainbow Trout following Immersion Infection: Organ Assay and Electron Microscopy

Abstract

Sequential spread of infectious hematopoietic necrosis virus (IHNV) to tissues of rainbow trout Oncorhynchus mykiss was examined following immersion infection with two different isolates of IHNV, a pathogenic strain and a nonpathogenic strain from rainbow trout. Virus strain 193–110 was highly pathogenic to 1-month-old rainbow trout and caused 100% mortality within 13 d, whereas strain RB-76 was much less virulent, causing 50% mortality by the 19th day. Virus titers of 1-month-old fingerling fish dying soon after infection were significantly higher than titers of those dying later. Assays of dissected tissues showed that gills of infected 2-month-old fingerlings contained virus as early as 16 and 20 h postinfection, with definite replication occurring at 48 h. The early presence of the virus in the gills followed shortly by appearance of the virus in the kidneys and spleen indicated that the virus spreads rapidly to the target organs. Virus was detected in many other organs at lower levels on the third day and increased to higher levels during the following days. Heart tissue had high titers later in the infection. When 4-month-old rainbow trout were infected with strain 193–110, the mortality was reduced and delayed, whereas those infected with strain RB-76 produced no mortality. Assays on the day of death of these older fingerlings infected with strain 193–110 revealed that fish dying soon after infection also had higher titers than those dying later. Electron microscopic examination offish organs showed the presence of typical IHNV particles budding off from various tissue cells of affected organs, including gill tissue. The destructive effect of the virus was particularly noticeable in the disarrangement of heart muscle organelles.     

Spatial and Temporal Variation of Whirling Disease Risk in Montana Spring Creeks and Rivers

Abstract

Spring creeks are important spawning and rearing areas for wild trout, but the stable flows, cool temperatures, and high nutrient levels that characterize these unique habitats may also make them highly susceptible to establishment and proliferation of the whirling disease pathogen Myxobolus cerebralis. We evaluated the spatial and temporal dynamics in whirling disease risk by using sentinel rainbow trout Oncorhynchus mykiss fry in nine different spring creeks and their conjoining rivers or reservoirs in Montana over a 20-month period. Whirling disease risk was high in five of the seven pathogen-positive spring creek study sites; at these sites, prevalence levels exceeded 90% and over 50% of sentinel fry had moderate to high infection severity scores. Spring creeks generally had higher disease prevalence and severity than paired river or reservoir sites. Fine sediment levels varied widely among springs creeks with high and low whirling disease risk, and we found no significant association between fine sediment level and infection severity. The low risk measured for some spring creeks was likely attributable to the pathogen invasion being in its early stages rather than to environmental characteristics limiting the severity of infection. High whirling disease risk occurred over a wide range of temperatures at spring creek sites (4.5–13°C) and river sites (1.7–12.5°C). There was an unusual seasonal cycle of infection in spring creeks, with peak infection levels occurring from late fall to early spring and declining to near zero in late spring to early fall. The low infection risk during spring suggests that spring-spawning trout would be at a low risk of infection, even in spring creeks with otherwise high disease severity. In contrast, fry of fall-spawning trout may be much more susceptible to infection in spring creek environments.

Received November 22, 2011; accepted May 7, 2012     

White Sturgeon as a Potential Vector of Infectious Hematopoietic Necrosis Virus

Abstract

Cell lines from white sturgeon Acipenser transmontanus were derived from peripheral blood cells, heart, and spleen. Incubated with infectious hematopoietic necrosis virus (IHNV) for 8 d at l5°C, these cell lines produced 0.7–53.2 plaque-forming units (PFU)/cell. Waterborne exposure of larval white sturgeons (60 d posthatch) to 10⁶ PFU/mL of IHNV resulted in 10% mortality 5–6 d postinfection, with virus concentrations consistently greater than 10⁵ PFU/g. A replicate group of larval white sturgeons that were sampled at different times post-IHNV exposure had no detectable virus at 24 h, but 72% of the fish had IHNV concentrations of 10²-10⁶ PFU/g when they were examined 2–9 d postinfection. Juvenile white sturgeons (mean weight, 35 g) immersed in or injected with IHNV exhibited no mortality, and virus was only detected immediately postexposure in just 25% of the fish tested. Juvenile white sturgeons fed either virus-free rainbow trout Oncorhynchus mykiss or dead IHNV-infected rainbow trout had no viable virus in their feces. Juvenile white sturgeons fed or exposed to IHNV failed to transmit the virus to cohabiting rainbow trout fry. These results suggest that IHNV can replicate in larval white sturgeons but presumably not in juveniles or adults. Virus neutralization activity was detected in serum from adult white sturgeons (4–6 years old) cultured with rainbow trout exposed to IHNV but not in white sturgeons kept in a pathogen-free environment and fed a manufactured diet. White sturgeon serum with IHNV-neutralizing activity was used to passively immunize rainbow trout, and it provided significant (P < 0.01) protection against IHNV challenge.     

Infectious Pancreatic Necrosis Virus: Transmission from Infectious to Susceptible Rainbow Trout Fry

Abstract

Fry of rainbow trout Oncorhynchus mykiss were exposed to serotype VR-299 of infectious pancreatic necrosis virus (IPNV) by using a standardized immersion challenge. In concurrent experiments, fish were monitored for 11 d for excretion of IPNV or monitored for 9 d for excretion and transmission of IPNV to susceptible rainbow trout fry. Immersion-challenged fish began excreting virus within 2 d after challenge. The rate of IPNV excretion per fish increased steadily from about day 4 to day 8 and then decreased. Virus concentrations in tissues of immersion-challenged fish increased exponentially. Susceptible fish became infected with IPNV within 4 d after being introduced to immersion-challenged fish (e.g., 2 d after the challenged fish began excreting virus). By 9 d, 84% of the susceptible fish were infected with IPNV.     

Ranking freshwater fish farms for the risk of pathogen introduction and spread

A semi-quantitative model is presented to rank freshwater rainbow trout farms within a country or region with regards to the risk of becoming infected and spreading a specified pathogen. The model was developed to support a risk-based surveillance scheme for notifiable salmonid pathogens. Routes of pathogen introduction and spread were identified through a process of expert consultation in a series of workshops. The routes were combined into themes (e.g. exposure via water, mechanical transmission). Themes were weighted based on expert opinion. Risk factors for each route were scored and combined into a theme score which was adjusted by the weight. The number of sources and consignments were used to assess introduction via live fish movements onto the farm. Biosecurity measures were scored to assess introduction on fomites. Upstream farms, wild fish and processing plants were included in assessing the likelihood of introduction by water. The scores for each theme were combined to give separate risk scores for introduction and spread. A matrix was used to combine these to give an overall risk score. A case study for viral haemorrhagic septicaemia is presented. Nine farms that represent a range of farming practices of rainbow trout farms in England and Wales are used as worked examples of the model. The model is suited to risk rank freshwater salmonid farms which are declared free of the pathogen(s) under consideration. The score allocated to a farm does not equate to a quantitative probability estimate of the farm to become infected or spread infection. Nevertheless, the method provides a transparent approach to ranking farms with regards to pathogen transmission risks. The output of the model at a regional or national level allows the allocation of surveillance effort to be risk based. It also provides fish farms with information on how they can reduce their risk score by improving biosecurity. The framework of the model can be applied to different production systems which may have other routes of disease spread. Further work is recommended to validate the allocated scores. Expert opinion was obtained through workshops, where the outputs from groups were single point estimates for relative weights of risks. More formal expert opinion elicitation methods could be used to capture variation in the experts' estimates and uncertainty and would provide data on which to simulate the model stochastically. The model can be downloaded (in Microsoft(?)-Excel format) from the Internet at: http://www.cefas.defra.gov.uk/6701.aspx.     

Risk factors for outbreaks of disease attributable to white sturgeon iridovirus and white sturgeon herpesvirus-2 at a commercial sturgeon farm

OBJECTIVE: To determine management, fish, and environmental risk factors for increased mortality and an increased proportion of runts for white sturgeon exposed to white sturgeon iridovirus (WSIV) and white sturgeon herpesvirus-2 (WSHV-2). ANIMALS: White sturgeon in 57 tanks at 1 farm and observations made for fish at another farm. PROCEDURE: A prospective cohort study was conducted. Data on mortality, proportion of runts, and potential risk factors were collected. Five fish from each tank were examined for WSIV and WSHV-2 via inoculation of susceptible cell lines and microscopic examination of stained tissue sections. An ANCOVA was used to evaluate effects of risk factors on mortality and proportion of runts. RESULTS: Major determinants of number of dead fish (natural logarithm [In]-transformed) were spawn, source (90% confidence interval [CI] for regression coefficient, 0.62 to 2.21), and stocking density (90% CI, 0.003 to 0.03). Main predictors of proportion of runts (In-transformed) were spawn, mortality incidence density (90% CI, 0.004 to 0.03), age (90% CI, -0.012 to -0.004), and the difference in weight between the largest and smallest nonrunt fish (90% CI, 0.0002 to 1.24). Additional observations indicated a possible protective effect attributable to previous exposure to the viruses. CONCLUSIONS AND CLINICAL RELEVANCE: Mortality and proportion of runts for white sturgeon after exposure to WSIV and WSHV-2 may be reduced for a farm at which the viruses are endemic by selection of specific broodstock, stocking with fish that survived outbreaks of viral disease, using all-in, all-out production, and decreasing stocking densities.     

Prevalence of Flavobacterium psychrophilum bacterial cells in farmed rainbow trout: Characterization of metallothionein A and interleukin1-β genes as markers overexpressed in spleen and kidney of diseased fish

The aim of the present study was to assess the prevalence of the flavobacteria within farmed trout and to quantify their bacterial burden. A total of 61 fish were sampled from seven farms, and were distributed in two groups: (1) visibly diseased fish suffering from the rainbow trout fry syndrome or the bacterial cold water disease caused by the bacteria Flavobacterium psychrophilum and (2) normally appearing fish. F. psychrophilum cells were titered by qPCR, targeting a specific area of the 16S rRNA gene in skin, muscle, gills, liver, spleen and kidney from all fish. The pathogen was detected in these organs whatever the health status, with titers ranging from 10⁴ to 6 × 10⁷ bacteria/g of tissue in normally appearing fish, thus showing they were bacterial carriers. Two organs allowed differentiation between diseased and normally appearing fish: spleen and kidney, with titers ranging from 10⁶ to 10⁷ bacteria/g of tissue in normally appearing fish vs 10¹¹ to 10¹² bacteria/g of tissue in diseased fish. No relationship was found between immunoglobulin M-like titer in plasma and health status. Gene expression analysis in fish organs revealed two genes that were markers of the bacterial infection: mt-a and il-1β genes encoding the metallothionein A and the interleukin1-β, respectively. These genes were both over-expressed in gills, liver, spleen and kidney of diseased fish. Four genes encoding immunity markers were down-regulated in spleen (a key organ implicated in immunity) of diseased fish: tgf-β, cd8-α, mhc2-β and igt, suggesting a weakened immune system in diseased fish.