Nonisotopic detection of Loma salmonae (microspora) in rainbow trout (Oncorhynchus mykiss) gills by in situ hybridization

Loma salmonae, a microsporidian parasite of salmonids of the genus Oncorhynchus, is a significant cause of economic loss in pen-reared chinook salmon (O. tschawytscha). Final stages of L. salmonae infections are easily recognized by the xenomas that form in the gills during sporogony. However, early prexenoma stages of infection (3 weeks or less after infection) are difficult to detect on histologic slides. An L. salmonae-specific single-stranded DNA probe labeled with digoxigenin was used to detect these prexenoma stages of L salmonae by in situ hybridization in experimentally infected rainbow trout. This method allows detection of the parasite in the gills only 2 weeks after infection, providing a sensitive and specific way of detecting L. salmonae during the early stages of infection.     

The Gill Pathogen Dermocystidium salmonis in Oregon Salmonids

Abstract

Intense infections of the gill pathogen Dermocystidium salmonis were associated with mortality of prespawning chinook salmon Oncorhynchus tshawytscha in several Oregon rivers in 1988. The occurrence of the pathogen in returning adult chinook salmon was monitored in several coastal Oregon stocks from 1989 to 1993. Although the prevalence of the pathogen was high in these fish (up to 66.6%), infection intensities were generally low, and no mortality attributable to D. salmonis was observed. In 1988, the pathogen was associated with a lethal epizootic among juvenile chinook salmon smolts at the Trask State Fish Hatchery near Tillamook, Oregon. Histological examination of gills from heavily infected fish revealed hyperplasia of gill epithelium and fusion of gill lamellae. When naturally infected smolts were transferred from fresh to salt water, the most heavily infected fish died within 10 d, and the number of D. salmonis cysts declined and disappeared from previously infected salmon after 21–42 d.     

Localization of the initial developmental stages of Loma salmonae in rainbow trout (Oncorhynchus mykiss)

The intracellular microsporidian parasite Loma salmonae affects salmonids of the genus Oncorhynchus and is a significant cause of economic losses in pen-reared Chinook salmon (O. tshawytscha) in British Columbia. Loma salmonae infection is easily recognized by the xenomas that form in the gills, but early stages of infection are difficult to detect in histologic sections. In situ hybridization (ISH), using an L. salmonae-specific digoxigenin-labeled single-stranded DNA probe, was used to detect the parasite during the early stages of infection. Loma salmonae was detected in the gut mucosal epithelium as early as 24 hours postexposure (PE), and it localized in the lamina propria of the intestine within 24 hours of infection. After the parasite was detected in the lamina propria, dividing merogonic stages in infected cells in the heart were detected by ISH as early as 2 days PE, providing the first evidence of parasitaemia and hematogenous distribution of this parasite in infected blood cells. The parasites inside the infected cells appeared to be undergoing merogony as they passed through the heart, indicating that proliferation may start at the site of infection, before the parasite arrives to the gills for their final developmental phase. This is the first time that L. salmonae passage through the intestinal wall and migration to the heart has been visualized; however, the identity of the cells harboring the parasite has yet to be determined.     

Experimental Transmission and Pathogenicity of a Viral Erythrocytic Infection in Sea Bass Dicentrarchus labrax

Abstract

The experimental transmission of a viral erythrocytic infection (VEI) in sea bass Dicentrarchus (=Morone) labrax of the Mediterranean is reported. Transmission procedures included the intraperitoneal inoculation of healthy fishes with blood and hematopoietic tissue extracts from VEI-affected fishes, direct contact between VEI-affected and unaffected fishes, and rearing of fishes in unfiltered, nonirradiated seawater that had been used for holding VEI-affected fishes. Successful transmission of the infection was achieved in all experimental conditions, as ascertained by the presence of inclusion bodies in Giemsa-stained blood cells and subsequently confirmed by indirect immunofluorescent staining. Transmission of VEI by the inoculation of filtered cell-free extracts from affected tissues confirmed not only the infectious character of the disease but also its viral etiology. Two different patterns of effects on the hematological variables were observed: an erythroblastic polycythemia occurred 3 months after infection, whereas a moderate anemia appeared after 5–6 months. Fish growth was also negatively affected by VEI. Sea bass infected with VET were found to be more susceptible to bacterial challenge infection with Vibrio anguillarum isolate AO 18 than were control fish; the V. anguillarum dose that was lethal to 50% of the test fish was lower for VEI-affected fish, and their cumulative mortality in the bacterial challenge was higher. The injection of cyclophosphamide produced immunosuppression in unaffected sea bass and an increase in the level of infection in VEI-affected fishes; these results suggest the role of the immunological status of the host sea bass in the development of VEI.     

Chinook Salmon Epizootics in Lake Michigan: Possible Contributing Factors and Management Implications

Abstract

Stability of the Lake Michigan fishery for chinook salmon Oncorhynchus tshawytscha at high levels became questionable after stocks declined dramatically following spring epizootics in which bacterial kidney disease (BKD) was a major factor. Initially stocked in 1967, favorable survival and growth of chinook salmon through the 1970s led to increases in abundance and in popularity with anglers. Returns of chinook salmon improved annually until the late–1980s, when, with little warning, spring epizootics reduced the abundance of adult salmon by 50% or more. Reduced abundance of alewives (Alosa pseudoharengus), coupled with an increase in chinook salmon density and heavy parasite infection rates were hypothesized to have reduced chinook salmon growth and fitness and to have increased their susceptibility to BKD. Evidence of slower growth exists and low food availability may be the stressor that triggered the epizootics. Chinook salmon were a major component of the economic development and subsequent hardship of the sportfishing industry on Lake Michigan. Sustaining the chinook salmon fishery at previous levels may require managing for high abundance of alewives, which may be inconsistent with overall fish community management goals. The future sustainability and role of chinook salmon needs to be reevaluated in the context of the entire Lake Michigan fish community.     

Influence of Infection with Renibacterium salmoninarum on Susceptibility of Juvenile Spring Chinook Salmon to Gas Bubble Trauma

Abstract

During experiments in our laboratory to assess the progression and severity of gas bubble trauma (GBT) in juvenile spring chinook salmon Oncorhynchus tshawytscha, we had the opportunity to assess the influence of Renibacterium salmoninarum (Rs), the causative agent of bacterial kidney disease, on the susceptibility of salmon to GBT. We exposed fish with an established infection of Rs to 120% total dissolved gas (TDG) for 96 h and monitored severity of GBT signs in the fins and gills, Rs infection level in kidneys by using an enzyme-linked immunosorbent assay (ELISA), and mortality. Mortality occurred rapidly after exposure to 120% TDG, with a LT20 (time necessary to kill 20% of the population) of about 37 h, which is at a minimum about 16% earlier than other bioassays we have conducted using fish that had no apparent signs of disease. Fish that died early (from 31 to 36 h and from 49 to 52 h) had significantly higher infection levels (mean ± SE ELISA absorbance = 1.532 ± 0.108) than fish that survived for 96 h (mean ± SE ELISA absorbance = 0.828 ± 0.137). Fish that died early also had a significantly greater number of gill filaments occluded with bubbles than those that survived 96 h. Conversely, fish that survived for 96 h had a significantly higher median fin severity ranking than those that died early. Our results indicate that fish with moderate to high levels of Rs infection are more vulnerable to the effects of dissolved gas supersaturation (DGS) and die sooner than fish with lower levels of Rs infection. However, there is a substantial amount of individual variation in susceptibility to the apparent cumulative effects of DGS and Rs infection. Collectively, our findings have important implications to programs designed to monitor the prevalence and severity of GBT in juvenile salmonids in areas like the Columbia River basin, and perhaps elsewhere.     

Protection of Chinook Salmon Smolts with Oral Doses of Erythromycin against Acute Challenges of Renibacterium salmoninarum

Abstract

We challenged duplicate groups of yearling smolts of chinook salmon Oncorhynchus tshawytscha held in seawater with an intraperitoneal inoculation of the kidney disease bacterium Renibacterium salmoninarum 1 d before and at intervals of 1, 11, and 29 d after a 21-d oral administration of erythromycin thiocyanate at 0.1 g/kg body weight per day. Most mortality attributable to bacterial kidney disease (BKD) in fish challenged with R. salmoninarum but not administered erythromycin occurred 2–3 weeks after challenge; the average survival 35 d after challenge was only 9%. Nearly 70% of the fish challenged 1 d before the erythromycin feeding were alive 35 d after the 21-d treatment, and more than 98% of the fish challenged the day after the 21-d erythromycin treatment survived a further 35 d. Fish challenged 29 d after the erythromycin treatment were not significantly protected against BKD. Chinook salmon that were not challenged with the bacterium but were injected with sterile phosphate-buffered saline and then fed a ration with erythromycin survived at a significantly higher rate than unchallenged fish injected with saline but not fed erythromycin. Fish that were not injected with saline or R. salmoninarum survived at a significantly higher rate than fish handled and injected with saline or pathogen. Because erythromycin protected fish challenged just before and immediately after treatment, the antibiotic should be useful early in an outbreak of BKD and as a prophylactic when stresses are expected.     

Infectious Hematopoietic Necrosis Virus (IHNV) in Coho Salmon

Abstract

Infectious hematopoietic necrosis virus (IHNV) causes important losses of chinook salmon Oncorhynchus tshawytscha, sockeye salmon Oncorhynchus nerka, and rainbow trout and steelhead Oncorhynchus mykiss on the west coast of North America. Although coho salmon Oncorhynchus kisutch are considered resistant to IHNV infection, the virus was detected in numerous adult coho salmon returning to Trinity River Hatchery, California, in 1985 and 1986. The virus was isolated from internal organs and ovarian fluids of these fish. Antigenic and structural polypeptides of the viruses were identical in adult coho and chinook salmon collected at the same location. Chinook salmon and rainbow trout alevins exhibited high degrees of susceptibility to IHNV obtained from adult coho and chinook salmon. Coho salmon alevins were resistant to both virus isolants.     

Influence of reduced feed ration on Lepeophtheirus salmonis infestation and inflammatory gene expression in juvenile pink salmon

The effect of reduced feed ration on infestation levels with the sea louse Lepeophtheirus salmonis and gene expression in juvenile pink salmon Oncorhynchus gorbuscha was tested in three laboratory trials. Body weight was significantly lower among fish on the reduced ration for 27, 34, or 65 d than fish on the full ration. Neither the prevalence nor the abundance of L. salmonis differed between fish on full and reduced rations at any time in any trial. In trial 2, sea louse rejection was delayed among fish on reduced rations; however, the parasite was ultimately rejected from all fish in this trial regardless of ration. Proinflammatory gene expression in salmon exposed to L. salmonis was modulated by reduced rations. There was a reduction in the expression of interleukin-8 in pink salmon on reduced rations 7 d after exposure but not 14 d after exposure. In contrast, the 7-d expression of interleukin-1 beta (IL-1beta) was reduced in exposed pink salmon regardless of ration. By day 14, however, expression of IL-1beta was increased in association with reduced rations among exposed salmon. Similarly, the expression of tumor necrosis factor alpha (TNFalpha) was increased 14 d after exposure among salmon on a reduced ration. There was no evidence that short-duration exposure of otherwise healthy juvenile pink salmon to a reduced ration affected susceptibility to L. salmonis. The expression data do not suggest an obvious mechanism of louse rejection; rather, they indicate that a more comprehensive suite of inflammatory pathways should be surveyed to better understand the early pink salmon response to L. salmonis.     

Preliminary investigation of a possible lung worm (Parafilaroides decorus), fish (Girella nigricans), and marine mammal (Callorhinus ursinus) cycle for San Miguel sea lion virus type 5

Colostrum-deprived neonatal Northern fur seal pups (Callorhinus ursinus) were exposed to San Miguel sea lion virus type 5 (SMSV-5) by feeding them fish (Girella nigricans) infected with virus or fish infected with both the sea lion lung worm larvae (Parafilaroides decorus) and virus. Virus infection was demonstrated in 8 of 9 pups, and 1 of these developed a vesicular lesion on the flipper. In this sequence, P decorus larvae exposed to SMSV-5 were fed to G nigricans held at 15 C in a salt water aquarium; 32 days later, these fish were killed, then fed to the fur seal pups. The vesicle developed 22 days subsequent to this and SMSV-5 was reisolated from the lesion. The SMSV-5 was shown to persist for at least 23 days in infected neonatal fur seals. Attempts to establish P decorus infection in Northern fur seal pups were apparently unsuccessful.     

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.     

Transmission of the microsporidian gill parasite, Loma salmonae

Since it was first reported in 1987 at a hatchery in British Columbia, Loma salmonae has become increasingly important as an emerging parasite affecting the Canadian salmonid aquaculture industry. L. salmonae causes Microsporidial Gill Disease of Salmon (MGDS) in farmed Pacific salmonids, Oncorhynchus spp., resulting in respiratory distress, secondary infections and high mortality rates. In the last decade, laboratory studies have identified key transmission factors for this disease and described the pathogenesis of MGDS. L. salmonae enters the host via the gut, where it injects sporoplasm into a host cell, which then migrates to the heart for a two-week merogony-like phase, followed by a macrophage-mediated transport of the parasite to the gill, with a final development stage of a spore-laden xenoma within the endothelial and pillar cells. Xenoma rupture triggers a cascade of inflammatory events leading to severe, persistent, and extensive proliferative branchitis. The development of robust and reliable experimental challenge models using several exposure methods in marine and freshwater environments with several fish hosts, is a primary reason for the success of scientific research surrounding L. salmonae. To date, demonstrated factors affecting MGDS transmission include host species, strain and size, the length of contact time between na?ve and infected fish, water temperature and flow rates.     

Pathogenesis of liver lesions caused by experimental infection with Piscirickettsia salmonis in juvenile Atlantic salmon, Salmo salar L.

Piscirickettsia salmonis, the etiologic agent of salmonid rickettsial septicemia (SRS), or piscirickettsiosis, causes substantial economic losses to the salmon industry. The pathogenesis of the disease has not been fully characterized. The aim of this study is to describe the hepatic lesions associated with experimental P. salmonis infection in Atlantic salmon juveniles. Fish were maintained in fresh water and inoculated intraperitoneally (IP), orally, or on the gill surface with P. salmonis. A group of uninfected fish was kept as control. Liver samples from 5 fish in each inoculated group and 3 controls were collected weekly and processed for histological and immunohistochemical examination. Thickening of the liver capsule by inflammatory cells was a characteristic histologic feature of IP inoculated fish. Three weeks post-IP inoculation, 8 fish had died and 2 fish were sampled. Histological changes at this time consisted of vasculitis, presence of fibrin thrombi, vacuolated hepatocytes and focal areas of necrosis. Leukocytes containing intracytoplasmic basophilic microorganisms were seen within hepatic sinusoids. Vasculitis and intracytoplasmic vacuoles were prominent features in fish inoculated orally and on the gill surface. The presence of P. salmonis within hepatocellular vacuoles, endothelial cells, and leucocytes was confirmed by immunohistochemistry. The intracellular location of P. salmonis and the vascular damage seen in infected fish are characteristic of rickettsial infections. Histological lesions induced by experimental infection with P. salmonis using the oral and gill surface routes were similar to those observed in natural outbreaks of piscirickettsiosis. The tropism of P. salmonis for endothelial cells explains the vascular lesions observed in SRS, whereas hepatic lesions are due to ischemic necrosis and direct injury by intracytoplasmic organisms.     

Demonstration of the Specificity of the Salmonid Hurnoral Response to Renibacterium salmoninarum with a Monoclonal Antibody against Salmonid Immunoglobulin

Abstract

The specificity of the antibody response of salmonids to Renibacterium salmoninarum antigens was demonstrated by western blotting techniques that utilized a monoclonal antibody against salmonid immunoglobulin. In this study, the specificity of the response in immunized chinook salmon Oncorhynchus tshawytscha was compared with the response in naturally infected chinook salmon and coho salmon O. kisutch, and immunized rabbits. The antibody response in immunized salmon and rabbits and the naturally infected fish was primarily against the 57–58kilodalton protein complex. In addition to recognizing these proteins in the extracellular fraction and whole-cell preparations, antibody from the immunized salmon and rabbits detected four proteins with lower molecular masses. Western blotting techniques allow identification of the specific antigens recognized and are a useful tool for comparing the immunogenicity of different R. salmoninarum preparations. Immunofluorescent techniques with whole bacteria were less sensitive than western blotting in detecting salmonid anti-R. salmoninarum antibody.     

Quantitation of immune response gene expression and cellular localisation of interleukin-1beta mRNA in Atlantic salmon, Salmo salar L., affected by amoebic gill disease (AGD)

The characterisation of selected immune response genes during amoebic gill disease (AGD) in Atlantic salmon, Salmo salar L., was performed using semi-quantitative RT-PCR, quantitative real-time RT-PCR (qRT-PCR), and in situ hybridisation (ISH). The immune response genes of interest were interleukin-1beta (IL-1beta), inducible nitric oxide synthase (iNOS), serum amyloid A (SAA), and serum amyloid P-like pentraxin (SAP). Atlantic salmon were inoculated with the ectoparasite Neoparamoeba sp., the causative agent of AGD, and gill, liver and anterior kidney tissue sampled at 0, 7 and 14 d post-inoculation (p.i.). Semi-quantitative RT-PCR was performed on the tissue samples to identify up/down-regulated mRNA expression relative to uninfected control fish and normalised to the housekeeping gene, beta-actin. Interleukin-1beta (IL-1beta) was the only immune response gene of those investigated whose mRNA was differentially regulated in any of the tissues and was found to be up-regulated in the gills by semi-quantitative RT-PCR. Increased gill IL-1beta mRNA expression was then accurately quantitated and confirmed using probe-based qRT-PCR. The cellular localisation of the IL-1beta mRNA expression in the gills of uninfected and infected fish was then determined by ISH using an IL-1beta-specific biotinylated cRNA probe. Expression of IL-1beta mRNA was localised to filament and lamellar epithelium pavement cells in gills of uninfected and infected Atlantic salmon. These data implicate the involvement of IL-1beta at the site of infection, the gills, of Atlantic salmon during AGD. This work supports previous studies that suggest IL-1beta is important in the regulation of the fish immune response to parasitic infection but additionally shows the cellular localisation of fish IL-1beta mRNA expression during infection.     

Infectious Salmon Anemia Virus (ISAV) in Brown Trout

Abstract

Infectious salmon anemia (ISA) is a viral disease of Atlantic salmon Salmo salar that have been exposed to seawater in fish farms or hatcheries. This disease was previously believed to be exclusively one of salmon. However, it has been shown that anadromous brown trout Salmo trutta may carry the ISA virus (ISAV). Propagation of the ISAV in brown trout without the trout's showing any gross clinical signs of disease could be a result of a longstanding host-pathogen relationship between the virus and brown trout. A brown trout population isolated from the sea during the last 5,000 years and expected to be naive to the virus was challenged. These fish did not develop any gross signs of disease, but a few ISAVs were present as late as 46 d postchallenge. It was also shown that the ISA virus was present in brown trout as late as 7 months after challenge.     

Effects of Practices Related to Catch-and-Release Angling on Mortality and Viral Transmission in Juvenile Largemouth Bass Infected with Largemouth Bass Virus

Abstract

Largemouth bass virus (LMBV; family Iridoviridae) has recently emerged as a causative agent in fish kills of largemouth bass Micropterus salmoides. Little is known about how the virus is transmitted or what factors predispose fish to mortality subsequent to infection. Concern has nevertheless arisen that activities related to recreational angling may affect transmission dynamics and may alter the susceptibility of infected fish to clinical disease. This study examined the separate effects of two angling-related factors on the susceptibility of juvenile largemouth bass to mortality from LMBV infection and on the transmission of LMBV from infected to uninfected fish. The first factor was hook-and-line angling. Infected fish that underwent a simulated angling treatment did not experience higher mortality or have higher viral loads in their tissues than those that were not angled. The second factor was direct contact between infected and uninfected fish, as would occur in live wells and holding tanks. The LMBV was transmitted from infected to uninfected fish through water, even when direct contact was prevented. Transmission of LMBV between infected and uninfected fish separated by a fenestrated barrier was nearly as efficient as LMBV transmission between infected and uninfected fish that were allowed direct contact. These results imply that angling itself may have only minimal effects on the survival of largemouth bass infected with LMBV but that angling-related practices that place infected and uninfected fish together in a limited water volume may facilitate viral transmission. Partitioning or cooling of live wells and holding tanks, as well as limiting their use, could reduce LMBV-associated mortality and viral transmission.     

Erythrocytic Inclusion Body Syndrome (EIBS) Infection of Chinook Salmon in Idaho

Abstract

Intracytoplasmic inclusions (0.8–2.0 μm in diameter) were detected in erythrocytes of yearling chinook salmon Oncorhynchus tshawytscha from the Rapid River Hatchery in central Idaho. Virions with a mean diameter of 74 nm were found both free and within membrane-bound vacuoles in the cytoplasm of erythrocytes. Clinical signs of affected fish included anemia, hypoproteinemia, splenomegaly, hemosiderin deposits in the spleen, and concurrent saprolegniosis. Low water temperatures may have been responsible for the reduction in mortality rate and the extended infection period observed during the winter. Attempts at demonstrating horizontal transmission and identifying a source of infection were unsuccessful. This is the first report of erythrocytic inclusion body syndrome (EIBS) among salmonid fish in Idaho.     

A New Eye Disease in Pen-Reared Chinook Salmon Caused by Metacestodes of Gilquinia squali (Trypanorhyncha)

Abstract

Eye infections by metacestodes of Gilquinia squali (Cestoda: Trypanorhyncha) were associated with mortality in chinook salmon Oncorhynchus tshawytscha reared in net-pens in seawater at two separate locations in British Columbia. Mortality associated with the disease was about 10% at one site and undetermined at the second site. The metacestode was found in the vitreous humor of moribund fish with eye lesions, whereas healthy fish from the same pen exhibited neither eye lesions nor the infection. The lesions were characterized by retinal folding and apparent retinal duplication, and cataractous changes; in severe cases, the cornea ruptured, and the lens was extruded. The spiny dogfish Squalus acanthias i s the definitive host for the cestode. Spiny dogfish were prevalent in and around the affected net-pen sites, thus providing coracidia to infect the first intermediate host, which is most likely a crustacean. Although the metacestode is common in the eyes of European whiting Merlangius merlangus in the northeast Atlantic region, this is the first report of the infection in a salmonid.     

Development and Evaluation of a Monoclonal-Antibody-Based Enzyme-Linked Immunosorbent Assay for the Diagnosis of Renibacterium salmoninarum Infection

Abstract

A monoclonal-antibody-based enzyme-linked immunosorbent assay (ELISA) was developed for the diagnosis of bacterial kidney disease (BKD). This ELISA can detect Renibacterium salmoninarum antigen at concentrations as low as 0.05–0.1 μg/mL. During the 1988–1989 spawning season, 60 coho salmon Oncorhynchus kisutch, 60 chinook salmon O. tshawytscha, and 60 steelhead O. mykiss (Great Lakes rainbow trout) were caught and screened for BKD with the developed ELISA and a direct fluorescent antibody technique (FAT). Serum agglutination titers for R. salmoninarum were measured to determine any relationship between presence of antigen (R. salmoninarum) and humoral antibody to R. salmoninarum. Twelve of the coho salmon (20%), 48 of the chinook salmon (80%), and 7 of the steelhead (11.7%) were BKD-positive according to the ELISA. Only one steelhead (1.7%) was BKD-positive by FAT, whereas none of the coho salmon or chinook salmon were BKD-positive. It was concluded that the monoclonal-antibodybased ELISA was more sensitive than FAT. Antibody titers of these asymptomatic fish were variable. There was no correlation between antigen level and antibody titer.