The Effects of Myxobolus cerebralis on the Physiological Performance of Whirling Disease Resistant and Susceptible Strains of Rainbow Trout

Abstract

The development of rainbow trout Oncorhynchus mykiss strains that are resistant to whirling disease has shown promise as a management tool for populations in areas where Myxobolus cerebralis is present. However, the physiological effects of the disease on characteristics necessary for fish survival in natural river conditions have not been tested in many of these strains. Five rainbow trout strains were evaluated for their swimming ability and growth characteristics in relation to M. cerebralis exposure: the resistant German rainbow trout (GR) strain (Hofer strain), the susceptible Colorado River rainbow trout (CRR) strain, and three intermediate (hybrid) strains (F₁ = GR × CRR; F₂ = F₁ × F₁; B₂ = backcross of F₁ × CRR). Three broad response patterns among strain and exposure were evident in our study. First, exposure metrics, growth performance, and swimming ability differed among strains. Second, exposure to the parasite did not necessarily produce differences in growth or swimming ability. Exposure to M. cerebralis did not affect batch weight for any strain, and critical swimming velocity did not differ between exposed and unexposed families. Third, although exposure did not necessarily affect growth or swimming ability, individuals that exhibited clinical deformities did show reduced growth and swimming performance; fish with clinical deformities were significantly smaller and had lower critical swimming velocities than exposed fish without clinical deformities. Research and management have focused on GR × CRR hybrid strains; however, given the performance of the GR strain in our study, it should not be discounted as a potential broodstock. Additional field trials comparing the GR and F₁ strains should be conducted before wholesale adoption of the GR strain to reestablish rainbow trout populations in Colorado.

Received September 9, 2010; accepted May 27, 2011     

Comparison of hatchery and field performance between a whirling-disease-resistant strain and the Ten Sleep strain of rainbow trout

A whirling-disease-resistant strain of rainbow trout Oncorhynchus mykiss (GRHL strain) derived from a backcross of an F1 hybrid of two strains (German strain x Harrison Lake strain) with German strain females, was compared with the Ten Sleep (TS) strain of rainbow trout. The GRHL strain had consistently superior growth and feed conversion in two consecutive hatchery trials. Hatching and mortality rates were similar between strains. Both strains were stocked into two Utah reservoirs (Hyrum, Porcupine), and a third, Causey Reservoir, was monitored as a control for seasonal variation in prevalence of Myxobolus cerebralis. A total of 1,323 salmonids captured by gill net in spring and fall sampling between 2006 and 2008 were tested for M. cerebralis via pepsin-trypsin digest methods. Only eight of these (< 1% per species) had clinical signs consistent with whirling disease. In both reservoirs, GRHL survived better than the TS and had higher growth rates. The prevalence of M. cerebralis was significantly lower for GRHL (18.1%) than TS (50.0%) in Porcupine Reservoir. In Hyrum Reservoir the trend was similar, but prevalence was lower and did not significantly differ between GRHL (9.6%) and TS (23.1%). For infected fish, no significant differences were observed between strains in myxospore counts in either Hyrum (GRHL = 911-28,244 spores/fish [spf], TS = 1,822-155,800 spf) or Porcupine (GRHL = 333-426,667spf, TS = 333-230,511 spf) reservoirs. Unmarked rainbow trout in both reservoirs had significantly higher myxospore counts than stocked fish of either strain. There were significant differences in M. cerebralis prevalence and myxospore loads among other naturally reproducing salmonids in the reservoirs. The trend in susceptibility was cutthroat trout Oncorhynchus clarkii > kokanee Oncorhynchus nerka > brown trout Salmo trutta. The GRHL performed well in both hatchery and field settings and is recommended for stocking programs.     

Myxobolus cerebralis infection in rainbow trout (Oncorhynchus mykiss) and brown trout (Salmo trutta) exposed under natural stream conditions.

From early April into mid-June 1977, sequential groups of juvenile rainbow trout (Oncorhynchus mykiss) and brown trout (Salmo trutta) were each exposed for 10 days to the parasite Myxobolus cerebralis by immersion in a stream inhabited by infected wild trout. Following incubation in a M. cerebralis-free facility, trout were subsequently killed, and heads and gill arches were examined by routine histologic methods. A grading scale to quantify lesion severity was developed and applied. Percentage infected, lesion severity scores, effects of water temperature and flow rates on percentage infected and lesion severity scores, and resulting pathology were determined for each species at each exposure period. The percentage of rainbow trout infected with M. cerebralis was significantly higher than the percentage of brown trout infected for each exposure period. The percentages of rainbow trout infected in exposure periods later in the calendar year were significantly higher than those in earlier periods. The percentages of brown trout infected were not significantly different among exposure periods. Overall average lesion severity scores were significantly higher in rainbow than in brown trout. Lesion severity scores in rainbow trout increased over time (a positive correlation with exposure period). Lesion severity scores were not significantly different for brown trout among exposure periods. A significant correlation existed between water temperature and percentage of rainbow trout infected; a significant correlation also existed between water temperature and lesion severity scores in rainbow trout. Similar correlations did not exist for percentage of brown trout infected or accompanying lesion severity scores. In rainbow trout, ventral calvarium was the most common site of M. cerebralis replication, followed by gill arches. In brown trout, lesions were virtually confined to gill arches. Early lesions consisted of foci of cartilage necrosis with small numbers of M. cerebralis developmental stages. More advanced lesions consisted of multifocal areas of cartilage necrosis with numerous M. cerebralis developmental stages and/or mature myxospores bordered and/or infiltrated by mono- and multinuclear leukocytes. Lesions in brown trout were smaller and had fewer associated leukocytes and M. cerebralis developmental stages and/or mature myxospores. Higher infection rates, lesion severity scores, and differences in lesion location in rainbow versus brown trout explain in part why numbers of rainbow but not brown trout have fallen in western rivers inhabited with M. cerebralis-infected trout.     

Comparison of Hatchery and Field Performance between a Whirling-Disease-Resistant Strain and the Ten Sleep Strain of Rainbow Trout

Abstract

A whirling-disease-resistant strain of rainbow trout Oncorhynchus mykiss (GRHL strain) derived from a backcross of an F1 hybrid of two strains (German strain × Harrison Lake strain) with German strain females, was compared with the Ten Sleep (TS) strain of rainbow trout. The GRHL strain had consistently superior growth and feed conversion in two consecutive hatchery trials. Hatching and mortality rates were similar between strains. Both strains were stocked into two Utah reservoirs (Hyrum, Porcupine), and a third, Causey Reservoir, was monitored as a control for seasonal variation in prevalence of Myxobolus cerebralis. A total of 1,323 salmonids captured by gill net in spring and fall sampling between 2006 and 2008 were tested for M. cerebralis via pepsin-trypsin digest methods. Only eight of these (<1% per species) had clinical signs consistent with whirling disease. In both reservoirs, GRHL survived better than the TS and had higher growth rates. The prevalence of M. cerebralis was significantly lower for GRHL (18.1%) than TS (50.0%) in Porcupine Reservoir. In Hyrum Reservoir the trend was similar, but prevalence was lower and did not significantly differ between GRHL (9.6%) and TS (23.1%). For infected fish, no significant differences were observed between strains in myxospore counts in either Hyrum (GRHL = 911–28,244 spores/fish [spf], TS = 1,822–155,800 spf) or Porcupine (GRHL = 333–426,667spf, TS = 333–230,511 spf) reservoirs. Unmarked rainbow trout in both reservoirs had significantly higher myxospore counts than stocked fish of either strain. There were significant differences in M. cerebralis prevalence and myxospore loads among other naturally reproducing salmonids in the reservoirs. The trend in susceptibility was cutthroat trout Oncorhynchus clarkii > kokanee Oncorhynchus nerka > brown trout Salmo trutta. The GRHL performed well in both hatchery and field settings and is recommended for stocking programs.

Received December 28, 2011; accepted February 2, 2012     

Extracellular virulence factors of fish Vibrio: relationships between toxic material, hemolysin, and proteolytic enzyme

Biological activities of cell-free culture filtrate of 3 virulent strains of fish Vibrio were examined to determine the relationship to the pathogenesis of fish vibriosis. Among the 3 strains examined, V anguillarum strains NCMB6 and NCMB571 produced hemolysin and protease, whereas V ordalii strain N7802 did not. Culture filtrate of strain NCMB571 were lethal to rainbow trout and produced a cytotoxic effect on fish cell line. Results revealed that the extracellular products may be involved in the pathogenesis of fish vibriosis.     

The Effects of Whirling Disease on Growth and Survival of Snake River Cutthroat and Colorado River Rainbow Trout Fingerlings

Abstract

Two sizes of fingerling Snake River cutthroat trout Oncorhynchus clarkii behnkei and Colorado River rainbow trout O. mykiss were raised at hatcheries testing negative for Myxobolus cerebralis and stocked into the Dolores and Cache la Poudre rivers from 1999 to 2001. Populations were resampled over a 2-year period to determine which species and size combination had the highest growth and survival rates. Fish were tested for M. cerebralis via polymerase chain reaction and pepsin?trypsin digest analyses. Growth and survival rates between the species and size groups were not significantly different in either river. In the Dolores River, annual survival for both species and sizes of fish combined ranged from 0.063 to 0.12. In the Cache la Poudre River, survival for both sizes of rainbow trout was 0.004; survival for cutthroat trout ranged from 0.182 to 0.53. Larger fish had higher growth rates than smaller fish, and cutthroat trout had higher rates than similar sizes of rainbow trout. In both rivers, a higher percentage of the rainbow trout sample was infected than in the cutthroat trout sample. Rainbow trout also had a higher mean number of spores per head than cutthroat trout, and small rainbow trout had higher spore counts than large rainbow trout. Survival rates for cutthroat trout in the Cache la Poudre River were the highest of any of the groups, suggesting a difference that is biologically significant. Raising fingerlings to sizes greater than 100 mm can improve poststocking survival. If rainbow trout are stocked into contaminated waters, raising fingerlings to a larger size does not appear to improve growth or survival rates. Stocking rainbow trout in the spring could maximize growth rates but will expose fish to greater triactinomyxon densities, resulting in higher intensities of infection.     

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.     

Evaluation of five diagnostic methods for the detection and quantification of Myxobolus cerebralis.

Diagnostic methods were used to identify and quantify Myxobolus cerebralis, a myxozoan parasite of salmonid fish. In this study, 7-week-old, pathogen-free rainbow trout (Oncorhynchus mykiss) were experimentally infected with M. cerebralis and at 7 months postinfection were evaluated with 5 diagnostic assays: 1) pepsin-trypsin digest (PTD) to detect and enumerate spores found in cranial cartilage, 2) 2 different histopathology grading scales that provide a numerical score for severity of microscopic lesions in the head, 3) a conventional single-round polymerase chain reaction (PCR), 4) a nested PCR assay, and 5) a newly developed quantitative real-time TaqMan PCR. There were no significant differences (P > 0.05) among the 5 diagnostic assays in distinguishing between experimentally infected and uninfected control fish. The 2 histopathology grading scales were highly correlated (P < 0.001) for assessment of microscopic lesion severity. Quantification of parasite levels in cranial tissues using PTD and real-time TaqMan PCR was significantly correlated r = 0.540 (P < 0.001). Lastly, 104 copies of the 18S rDNA gene are present in the M. cerebralis genome, a feature that makes this gene an excellent target for PCR-based diagnostic assays. Also, 2 copies of the insulin growth factor-I gene are found in the rainbow trout genome, whose detection can serve both as an internal quality control for amplifiable DNA and as a basis to quantify pathogen genome equivalents present in quantitative PCR assays.     

Detection of early stages of Myxobolus cerebralis in fin clips from rainbow trout (Orynchus mykiss).

A nested polymerase chain reaction (PCR) assay was used to detect early stages of Myxobolus cerebralis in caudal and adipose fin samples from rainbow trout (RT). To determine sensitivity, groups of 10 RT were exposed to 2,000 M. cerebralis triactinomyxons/fish for 1 hour at 15 degrees C and subsequently moved to clean recirculating water. Fish were held for 2 and 6 hours and 1, 2, 3, 5, 7, 10, 30, and 60 days before sampling by nonlethal fin biopsy. Nested PCR performed on fin clips showed that M. cerebralis DNA was detected in caudal fin tissue in 100% of fish up to 5 days postexposure. At days 7 and 10 postexposure, 80% of fish were positive, and at 60 days postexposure, 60% of fish were positive using this technique. Conversely, testing on adipose fin clips proved less sensitive, as positive fish dropped from 80% at day 7 to below 20% at day 10 postinfection. Since detection of M. cerebralis infection using caudal fin samples coupled with nested PCR is an effective method for detection of early parasite stages, use of this technique provides for accurate, nonlethal testing.     

Distribution of Myxobolus cerebralis in Salmonid Fishes in Montana

Abstract

Over an approximately 2-year period, 20,974 fish (trout and other salmonid species) from 230 separate waters (creeks, rivers, lakes, reservoirs, ponds, hatcheries, and irrigation ditches) within 21 of the 22 major drainages in Montana were examined for Myxobolus cerebralis. Nine of the major river drainages have waters containing infected fish: Beaverhead, Big Hole, Blackfoot, Clark Fork above the Bitterroot River, Flathead below the south fork of the Flathead River, Jefferson, Madison, Missouri above the Marias River, and Yellowstone above the Bighorn River. The Beaverhead, Clark Fork above the Bitterroot River, Jefferson, Madison, and Missouri above the Marias River have the greatest number of waters containing fish infected with M. cerebralis. Comparisons of infection levels (number of pooled samples that contain fish infected with M. cerebralis) between species among these drainages show significantly lower levels of infection in brown trout Salmo trutta and rainbow trout Oncorhynchus mykiss in the Missouri above the Marias River drainage and significantly higher levels of infection of rainbow trout in the Jefferson. Comparisons of differences in infection levels between drainages among species show that, in the Beaverhead, Clark Fork above the Bitterroot River, and Madison, infection levels in brown trout are significantly higher than in rainbow trout. This is partially attributed to losses of juvenile rainbow trout because of M. cerebralis infection, leading to biased samples. Histopathologic studies showed lesions were consistently less severe in brown trout than other species and occurred in a different location (gill arches versus ventral calvarium). In six of the nine affected drainages (Beaverhead, Blackfoot, Clark Fork above the Bitterroot River, Flathead below the South Fork, Jefferson, and Madison), infected fish were found at or near the time that intensive sampling was initiated in the spring of 1995. In the three remaining affected drainages (Missouri above the Marias River, Yellowstone above the Bighorn River, and the Big Hole), infected fish were not identified until at least 15 months after the initiation of widespread testing. This indicates that in the first six drainages listed above, the infection was well established prior to 1995 but spread into the last three drainages in the ensuing months. Methods of transmission and the sources of infection are unknown, although the absence of infected fish in state, private, and federal hatcheries in Montana indicates hatchery fish from these sources are not likely to be responsible.     

Comparison of Susceptibility of Five Cutthroat Trout Strains to Myxobolus cerebralis Infection

Abstract

Susceptibility to infection by the myxosporean parasite Myxobolus cerebralis was compared among strains of cutthroat trout Oncorhynchus clarki in two separate exposure tests in the laboratory. In both tests, each strain was exposed to 1,000 triactinomyxons/fish for 2 h in 8.0 L of water. In the first test, three strains of 10-week-old cutthroat trout were compared: two strains of Bonneville cutthroat trout O. c. utah (Bear Lake and southern Bonneville strains) and Yellowstone cutthroat trout O. c. bouvieri. In the second test, these strains plus Snake River fine-spotted cutthroat trout O. c. subsp. and Colorado River cutthroat trout O. c. pleuriticus were exposed at either 5 or 10 weeks of age. The prevalence of the M. cerebralis infection was determined by single-round polymerase chain reaction (PCR) assay 5 weeks after exposure. In the first test, the prevalence was significantly lower in the Bear Lake strain of Bonneville cutthroat trout (78.5%) than in the Yellowstone (97.8%) or southern Bonneville (100%) strains when exposed at 10 weeks of age. In the second test, the Bear Lake strain also had significantly lower infection rates after exposure at 5 (54%) or 10 weeks (82%) of age than the other four strains, which did not differ from each other (94–100%). The severity of the infection was also significantly reduced in Bear Lake Bonneville cutthroat trout, as suggested by the strength of the product of the single-round PCR assay. These results suggest that intraspecific differences in susceptibility to M. cerebralis infection exist, further supporting the need to maintain the genetic diversity among subspecies and geographic variants of cutthroat trout.     

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.     

Potential for dissemination of the nonnative salmonid parasite Myxobolus cerebralis in Alaska

Myxobolus cerebralis, the myxozoan parasite responsible for whirling disease in salmonids, was first introduced into the United States in 1958 and has since spread across the country, causing severe declines in wild trout populations in the intermountain western United States. The recent detection of the parasite in Alaska is further evidence of the species' capability to invade and colonize new habitat. This study qualitatively assesses the risk of further spread and establishment of M. cerebralis in Alaska. We examine four potential routes of dissemination: human movement of fish, natural dispersal by salmonid predators and straying salmon, recreational activities, and commercial seafood processing. Potential for establishment was evaluated by examining water temperatures, spatial and temporal overlap of hosts, and the distribution and genetic composition of the oligochaete host, Tubifex tubifex. The most likely pathway of M. cerebralis transport in Alaska is human movement of fish by stocking. The extent of M. cerebralis infection in Alaskan salmonid populations is unknown, but if the parasite becomes dispersed, conditions are appropriate for establishment and propagation of the parasite life cycle in areas of south-central Alaska. The probability of further establishment is greatest in Ship Creek, where the abundance of susceptible T. tubifex, the presence of susceptible rainbow trout Oncorhynchus mykiss, and the proximity of this system to the known area of infection make conditions particularly suitable for spread of the parasite.     

Pathological effects of Arcobacter cryaerophilus infection in rainbow trout (Oncorhynchus mykiss Walbaum)

Arcobacter cryaerophilus was isolated from naturally infected rainbow trout (Oncorhynchus mykiss Walbaum), and its pathogenicity was tested by intramuscular injection into 40 healthy 1-year-old rainbow trout at 16 degrees C. The lethal dosage of 50% end point (LD50) for A. cryaerophilus was calculated 2.25 x 10(4) viable cells. Experimental infection caused deaths with gross clinical abnormalities such as degenerated opercula and gills, liver damage, haemorrhagic kidney and serous fluid in swollen intestines. The counts of A. cryaerophilus in kidney, liver and gills of experimentally infected fish ranged from 1.59 x 10(10) colony forming units (cfu)/g to 7.41 x 10(12) cfu/g. The means of erythrocyte (RBC) count, haematocrit level, serum cholesterol, triglyceride, albumin and total protein concentrations in the blood of the experimentally infected rainbow trout group were significantly lower than in the healthy fish. Leukocyte (WBC) counts of the experimentally infected rainbow trout were significantly higher than those of healthy fish. The present work shows that the selected blood characteristics may be good indicators of response to infections in rainbow trout.     

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.     

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.     

Susceptibility of Selected Inland Salmonids to Experimentally Induced Infections with Myxobolus cerebralis,the Causative Agent of Whirling Disease

Abstract

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 10⁶, whereas more resistant species such as bull trout had 10⁴ spores; no spores were found among Arctic grayling at 5 months postexposure.     

Safety and efficacy of an inactivated vaccine against Lactococcus garvieae in rainbow trout (Oncorhynchus mykiss)

We studied the safety and efficacy of an inactivated vaccine (Ichtiovac-Lg) against Lactococcus garvieae in rainbow trout (Oncorhynchus mykiss). In an initial dose-response experiment to test safety, we injected 50 rainbow trout weighing 30-40 g with a double dose of vaccine (0.2 ml) intraperitoneally. We observed these fish three times a day until day 50 post-vaccination when they were killed to evaluate visceral reactions, adhesions and intraperitoneal absorption. Survival was 100% in both the treatment and control groups and no significant differences were found in percentage of severe adhesions and pigmentation of peritonea and viscera. In a second trial, we injected 50 rainbow trout weighing 30-40 g with 0.1 ml of vaccine and a control group was injected with 0.1 ml of PBS intraperitoneally. On day 29 post-vaccination, both groups were challenged by intraperitoneal injection with 0.1 ml of a virulent heterologous strain of L. garvieae at 3 x 10(6) cfu ml(-1) and fish were observed for a further 21 days. At the end of the experiment, the survivals of the vaccinated fish and control group were 94 and 4%, respectively.     

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.     

Phagocytic activity of macrophages of rainbow trout against Vibrio anguillarum and the opsonising effect of antibody and complement

The phagocytosis of Vibrio anguillarum by peritoneal macrophages from normal rainbow trout was enhanced by antibody and complement. Treatment of either macrophages or the bacteria by antibody also enhanced opsonisation. Five weeks after immunisation with V anguillarum, the phagocytic activity of macrophages from rainbow trout was increased significantly compared with the activity of those from unvaccinated fish. Although agglutinin titres did not increase until three weeks after immunisation, seven out of 10 fish challenged one week after immunisation survived, indicating that immunised fish had developed resistance to vibrio infection before significant levels of antibody or phagocytic activity became detectable.