Antibodies against Discocotyle sagittata (Monogenea) in farmed trout

The relationship between Discocotyle sagittata intensities and host length, weight and specific anti-parasite antibody titres was studied in 3 year-classes of farmed rainbow trout Oncorhynchus mykiss and brown trout Salmo trutta at the end of the annual transmission cycle. Antibody titres were significantly higher in infected farmed fish than in naive controls, indicating that infection elicits immunoglobulin production. No correlation was found between host size and parasite burdens, nor between infection intensities and antibody titres.     

Immunization of rainbow trout Oncorhynchus mykiss against Discocotyle sagiffata (Monogenea)

Rainbow trout Oncorhynchus mykiss were injected intraperitoneally with 2 different Discocotyle sagittata extracts dissolved in PBS and subsequently exposed to controlled infection. Immunization resulted in significantly reduced (p < 0.0001) worm intensities in > 50% of vaccinated fish (response arbitrarily defined as parasite burdens < mean control intensity - 1 SD), irrespective of the immunization regime (different parasite extracts, dosing and application schedules) employed. The protective effect of worm extract applied in Freund's complete adjuvant (FCA) did not differ significantly from extract given in PBS. Vaccination with embryonated parasite eggs extract and with FCA alone did not result in partial immunity, suggesting the observed protective effect is specific. Immunized fish had significantly higher specific antibody titres at the time of dissection (as determined by ELISA) than both naive and control fish. Overall, a significant negative correlation was found between antibody titres and worm burdens, suggesting immunoglobulins are implicated in mediating partial immunity. Western blot tests indicated the 2 different worm extracts used to immunize fish share antigens, but each one primarily induced recognition of a distinct band (30 and 38 kDa). Immunization seems to promote a shift between 2 equilibria, rather than progressively increasing protection. This would explain why boosting did not increase immunity, and why 2 different extracts primarily inducing recognition of 2 distinct antigens provide similar degrees of protection. Although several other non-specific and cellular factors are likely to be involved in controlling parasite numbers, it cannot be excluded that antibodies could be involved in mediating the observed partial immunity.     

Evidence of complement-mediated killing of Discocotyle sagittata (Platyhelminthes, Monogenea) oncomiracidia

Discocotyle sagittata oncomiracidia were rapidly killed when incubated in na?ve plasma and immune sera from both rainbow trout (Oncorhynchus mykiss) and brown trout (Salmo trutta), the killing proceeding at a faster rate with blood material from the latter fish species. The lethal activity of na?ve plasma and immune sera was comparable. This was abolished after incubation at 45 degrees C for 30 min and by the addition of EDTA but not EGTA supplemented with Mg(2+), indicating that complement acting via the alternative pathway is responsible for the parasiticidal effect observed. Scanning electron micrographs showed varying degrees of surface disruption in larvae exposed to fish plasma, suggesting that complement acts by breaching the oncomiracidial tegument. Control (untreated) oncomiracidia showed no damage. Ultrastructural damage was more extensive in oncomiracidia exposed to brown trout plasma than to rainbow trout plasma for equal periods, suggesting that the complement cascade may be involved in mediating host susceptibility.     

Susceptibility of brown trout to Gyrodactylus salaris (Monogenea) under experimental conditions

Brown trout ( Salmo trutta ) from anadromous River Lierelva, resident Lake Tunhovd, and resident Nordmarka stocks were exposed to Gyrodactylus salaris -infected salmon parr. The brown trout were fed pellets before the experiments, except for one group of the Nordmarka stock which was starved for 19 days before the experiments. The mean number of parasites declined directly and rapidly post infection for all groups of trout. There were no pronounced differences in resistance between the anadromous and the resident stocks. G. salaris infections tended to persist longer on starved than on fed trout of the Nordmarka stock. The maximum parasite persistence on trout was 50 days, and as parasite numbers increased on some fish parasite reproduction must have occurred on those trout. However, the limited susceptibility and marked innate resistance of trout to G. salaris establishment, development and reproduction, suggest parasite metapopulations will not survive on this species. Nevertheless, trout may still play a role in the dispersal of G. salaris within and between rivers.     

Comparative susceptibility of rainbow trout Oncorhynchus mykiss and brown trout Salmo trutta to Myxobolus cerebralis, the cause of salmonid whirling disease.

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

Comparative susceptibility of Atlantic salmon, lake trout and rainbow trout to Myxobolus cerebralis in controlled laboratory exposures

The susceptibility of lake trout Salvelinus namaycush, rainbow trout Oncorhynchus mykiss and Atlantic salmon Salmo salar to Myxobolus cerebralis, the causative agent of whirling disease, was compared in controlled laboratory exposures. A total of 450 (225 for each dose) fry for each species were exposed to a low (200 spores per fish) or high (2000 spores per fish) dose of the infective triactinomyxon. At 22 wk post-exposure, 60 fish from each group, as well as controls for each species, were examined for clinical signs (whirling behavior, blacktail, deformed heads and skeletal deformities), microscopic lesions, and presence of spores. Rainbow trout were highly susceptible to infection, with 100% being positive for spores and with microscopic pathological changes in both exposure groups. Rainbow trout were the only species to show whirling behavior and blacktail. Atlantic salmon were less susceptible, with only 44 and 61% being positive for spores, respectively, in the low and high dose groups, while 68 and 75%, respectively, had microscopic pathology associated with cartilage damage. Rainbow trout heads contained mean spore concentrations of 2.2 (low dose) or 4.0 (high dose) x 10(6) spores g tissue(-1). The means for positive Atlantic salmon (not including zero values) were 1.7 (low) and 7.4 (high) x 10(4) spores g tissue(-1). Lake trout showed no clinical signs of infection, were negative for spores in both groups and showed no histopathological signs of M. cerebralis infection.     

Variability in swimming performance and underlying physiology in rainbow trout (Oncorhynchus mykiss) and brown trout (Salmo trutta)

We investigated intra- and interspecific variation in swimming performance and related physiological parameters in two members of the salmonid family. For our comparisons, we sourced juvenile brown trout (Salmo trutta) and rainbow trout (Oncorhynchus mykiss) from one hatchery and a second strain of rainbow trout from another. The hatcheries maintain genetic stocks obtained several decades ago from very different environments. We tested competing hypotheses: that there would be greater interspecific (across species) variation or that there would be greater intraspecific (within species) variation, owing to regional adaptations. To test these hypotheses, individual and small schools of five fish were taken to fatigue using the critical swimming speed test (U(crit)), and three post-exercise physiological metrics, packed red cell volume (hematocrit), citrate synthase and lactate dehydrogenase activity, were assessed. The majority of the results in swimming performance and hematocrit support that intraspecific variation was greater than interspecific variation, i.e. the location had a stronger effect than did genus. Variation in lactate dehydrogenase activity supported neither intra- nor interspecific variation as determining factors. In sum, our findings suggest that the performance of different species of salmonids from the same locale can be more similar than those of the same species from different areas.     

Comparative diets and foraging strategies of subyearling Atlantic salmon,brown trout,and rainbow trout during winter

Over‐winter survival of salmonids in streams is thought to be an important population regulation mechanism. Yet because of the difficulty of conducting field studies due to adverse weather or ice conditions, compared to other seasons, salmonid ecology during winter is least understood. Consequently, we sought to examine interspecific feeding associations of an important salmonid stream assemblage in the Lake Ontario watershed during winter. The diets of Atlantic salmon (Salmo salar) parr, brown trout (S. trutta) parr, and rainbow trout (Oncorhynchus mykiss) parr were significantly different in February but not in March. Salmonid diets differed from the benthos and the drift during both months. Dipterans (chironomids, simuliids, and tipulids) and ephemerellids were the major prey taxa consumed. All three species fed more heavily on prey items from the benthos than from the drift. The diet of Atlantic salmon had the highest similarity to the benthos whereas the diet of brown trout had the lowest similarity to the drift. All three salmonid species generally selected ephemerellids, limnephilids, and chironomids and avoided elmids. These winter feeding observations are the first reported for this specific salmonid assemblage and will help managers better understand interspecific associations during this critical period.     

Triiodothyronine is needed for the acclimatization of brown trout (Salmo trutta) or rainbow trout (Oncorhynchus mykiss) to seawater]

Brown and rainbow trout, held in freshwater at 13 +/- 1 degrees, were injected, every 3 days, with iopanoic acid (IOP: 5 mg/100 g body wt), an inhibitor of deiodination of thyroxine (T4) to triiodothyronine (T3). One group of IOP-treated rainbow trout was immersed in T3 (20 micrograms/l water). In IOP trout, plasma T3 fell to very low levels by day 7, while changes in T4 levels were less marked. In IOP + T3 trout plasma T3 increased fivefold, plasma T4 being unchanged. No mortality occurred and plasma osmolarity (OP) was not altered by any treatment. After direct transfer to seawater (30/1000), IOP trout were unable to acclimate to salinity: all died within 2 or 3 days, while the survival at day 3 was 100% in control brown trout and 45 and 74% in control and IOP + T3 rainbow trout respectively. OP increased more in IOP and less in IOP + T3 than in controls. There was a significant inverse correlation between T3, but not T4, plasma level, at the time of transfer and the OP 1 day later. In conclusion, although T3 does not play a significant role in osmoregulation in freshwater, T3 and therefore the deiodination of T4 into T3, were required for the development of hypo-osmoregulatory capacity involved in acclimation of trout to seawater.     

Demonstrating freedom from Gyrodactylus salaris (Monogenea: Gyrodactylidae) in farmed rainbow trout Oncorhynchus mykiss

This paper describes an approach to demonstrate freedom of individual rainbow trout farms from Gyrodactylus salaris Malmberg, 1957. The infection status of individual farms is relevant should G. salaris be introduced into a country or zone previously known to be free of the parasite. Trade from farms where G. salaris may have been introduced would be restricted until freedom had been demonstrated. Cage, fish and parasite sample sizes were calculated based on the minimum detectable prevalence (P*), test characteristics, population size, and Type I and II errors. Between 5 and 23 cages per farm would need to be sampled to demonstrate freedom at a cage level P* of 10%. The number of fish sampled per cage depended mainly on the test sensitivity (probability of correctly identifying an infected fish). Assuming a test sensitivity of 99% at the fish level, 59 fish per cage are needed (P* = 5%). Since G. salaris may exist in mixed infection with G. derjavini, testing a sample of gyrodactylid parasites may not result in the parasite being detected when present. Test sensitivity at the fish level depends on the number of gyrodactylids on the fish, the proportion of which are G. salaris and the number examined. Assuming a P* of 5% (i.e. G. salaris are at least 5% of the gyrodactylid population), between 20 and 73 parasites per fish would need to be sampled (depending on abundance) to maintain the Type I error at 0.01 (thus a fish level test sensitivity of 99%). This work identifies the critical information, and further research, needed to assess freedom from G. salaris with a known level of confidence; this is essential to provide a sound scientific basis for decision-making about disease control measures.     

Habitat shifts in rainbow trout: competitive influences of brown trout

Summary We compared habitat use by rainbow trout sympatric (three streams) and allopatric (two streams) with brown trout to determine whether competition occurred between these two species in the southern Appalachian Mountains. We measured water depth, water velocity, substrate, distance to overhead vegetation, sunlight, and surface turbulence both where we collected trout and for the streams in general. This enabled us to separate the effects of habitat availability from possible competitive effects. The results provided strong evidence for asymmetrical interspecific competition. Habitat use varied significantly between allopatric and sympatric rainbow trout in 68% of the comparisons made. Portions of some differences refelected differences in habitats available in the several streams. However, for all habitat variables measured except sunlight, rainbow trout used their preferred habitats less in sympatry with brown trout than in allopatry if brown trout also preferred the same habitats. Multivariate analysis indicated that water velocity and its correlates (substrate particle size and surface turbulence) were the most critical habitat variables in the interaction between the species, cover in the form of shade and close overhead vegetation was second most important, and water depth was least important.     

Comparative susceptibility of two different genetic types of tilapia to Neobenedenia sp. (Monogenea)

Two different genetic types of tilapia, Mozambique tilapia Oreochromis mossambicus (MT), and Pargo-UNAM (PU; a synthetic hybrid whose genetic composition is 50% Florida red tilapia, 25% Rocky Mountain tilapia, and 25% red variant Oreochromis niloticus), were acclimatized to salinity and exposed to seawater from the Gulf of Mexico off the port of Veracruz, Mexico. Both fish types were infected by the monogenean ectoparasite Neobenedenia sp. and were killed within 2 to 3 wk. A crude worm extract was prepared from whole specimens collected during the original outbreak and used to immunize naive hosts of the same 2 types of tilapia. Immunized fish were then exposed to seawater, which resulted in Neobenedenia sp. infection. Immunization did not confer any protection against Neobenedenia sp. infection. However, the experiment enabled detailed analysis of the dynamics of infection and comparison of the effects of the parasite on the 2 host types. Although both tilapia types exhibited similar resistance to infection (as they harbored similar parasite burdens in the early phase of infection), PU is less tolerant to Neobenedenia sp., as a mean parasite abundance of ca. 50 worms fish-' killed all hosts within a fortnight, while 22% of MT survived up to 3 wk, harboring a mean parasite abundance of ca. 900 worms fish-'. Our results suggest that, as reported elsewhere, Neobenedenia sp. could negatively affect mariculture off the Mexican coast of the Gulf of Mexico.     

Active biomonitoring with brown trout and rainbow trout in diluted sewage plant effluents

Brown trout Salmo trutta populations of numerous Swiss rivers are declining. Sewage plant effluents are discussed as a possible cause. To investigate the influence of sewage plant effluents, brown trout as well as rainbow trout Oncorhynchus mykiss were exposed to 10% diluted waste water over a period of 12 months. The effects were compared to those on trout kept in commercial tap water. The mortality rate was low and no pathogenic bacteria or viruses were recorded in exposed and tap-water animals. Parasitological examination revealed a mild infestation with Gryodactylus sp. in all groups. Macroscopically and histologically, only minor changes in gills, skin, and kidney of exposed animals were found when compared to fish kept in tap water. Degenerative and inflammatory reactions in the liver of exposed animals were the most prominent findings. Several brown trout caught in the River Langete showed marked proliferative, degenerative and inflammatory lesions of gills, liver, and kidney. The results do not suggest that waste-water effects would explain the decrease of fish populations. However, it is conceivable that the effluents in combination with other factors in the river enhance the development of changes.     

Plasma cortisol concentrations before and after social stress in rainbow trout and brown trout

Two related experiments examined the relationship between plasma cortisol concentrations and the development of social hierarchies in fish. In the first, rainbow trout, Oncorhynchus mykiss, and brown trout, Salmo trutta, were observed for dominance interactions when confined within single-species pairs for 4, 48, or 168 h. Subordinate members of a pair exhibited significantly higher cortisol concentrations than dominant and single fish, but the pattern of cortisol elevation differed between the two species, being quicker to rise and increasing to a higher level in rainbow trout. Cortisol concentrations were correlated with behavioural measurements; the more subordinate the behaviour exhibited by a fish, the higher its cortisol concentration. Social stress was a chronic stressor, and no acclimation to social status occurred during the week. In the second experiment, measurements of plasma cortisol were made before pairing of rainbow trout and then after 48 h of confinement in pairs. Subordinate fish demonstrated significantly higher concentrations of plasma cortisol both before and after social stress. It therefore appears that in addition to cortisol being elevated during periods of social stress, an association may exist between initial cortisol levels and the likelihood of a fish becoming subordinate.     

Wide range of susceptibility to rhabdoviruses in homozygous clones of rainbow trout

Inbred lines differentially susceptible to diseases are a powerful tool to get insights into the mechanisms of genetic resistance to pathogens. In fish, chromosome manipulation techniques allow a quick production of such homozygous lines. Using gynogenesis, we produced nine homozygous clones of rainbow trout from a domestic population (INRA Sy strain). We examined the variability between clones for resistance to two rhabdoviruses, the viral haemorrhagic septicaemia virus (VHSV) and the infectious haematopoietic necrosis virus (IHNV). Intraperitoneal injections and waterborne infections were performed in parallel for both viruses. No survival was recorded after intraperitoneal injection of VHSV or IHNV, indicating that fish from all clones were fully susceptible to both viruses by this route of infection. In contrast, the different clones showed a wide range of survival frequency after waterborne infection. The resistance levels to VHSV ranged from 0 to 99% and resistance was not abrogated when resistant and sensitive animals were mixed and subjected to waterborne infection. VHSV was recovered from 10% of resistant fish after waterborne infection, confirming that virus replication was possible in this context but effective only in a low proportion of the population. The different clones also exhibited a wide range of survival (0-68%) after a waterborne infection with IHNV. Although VHSV-resistant clones were not fully resistant to IHNV, the susceptibility to IHNV and VHSV tended to be correlated, suggesting that non-specific mechanisms common to both viruses were involved.     

Immunohistochemical localization of rainbow trout ladderlectin and intelectin in healthy and infected rainbow trout (Oncorhynchus mykiss)

In the present study, the pattern of immuno-reactive ladderlectin and intelectin in healthy rainbow trout is compared to rainbow trout infected with a variety of infectious agents. In healthy rainbow trout, both proteins were localized to individual epithelial cells of the gill and intestine and both proteins were clearly demonstrated within cytoplasmic granules of polymorphonuclear leucocytes and macrophages/monocytes found in blood vessels, hepatic sinusoids, renal interstitium, mucosal epithelium and submucosa of normal intestine. In tissue from infected rainbow trout, there was an overall relative increase in both lectins compared to healthy fish and both proteins were detected in extra-cellular spaces surrounding bacteria, fungi and protozoa. Increased distribution and density of both RTLL and RTInt was demonstrated along mucosal surfaces and within inflammatory leucocytes in infected tissues and immune related organs. These findings represent one of the few examples of in vivo association of defence lectins and infectious agents.     

Monoclonal antibodies to lymphocytes of rainbow trout (Oncorhynchus mykiss)

Monoclonal antibodies (Mabs) to lymphocytes of rainbow trout have been developed by immunisation with synthetic peptides, prepared from selected parts of the alpha- and beta-gene sequences of the T-cell receptor (TCR). Mab 1C2 (TCR beta immunisation) identified lymphocytes in blood (11%), spleen (18%) and in thymus (9%) in flow cytometry analysis (FCM). Immune complexes of lymphocytes coupled to Mab 1C2 was used for further immunisations resulting in numerous supernatants reactive with lymphocytes in FCM, of which Mabs 7A5 and 8H4 were selected for further characterisation. Mab 7A5 identified 31% of lymphocytes in blood and 9% in the spleen. Mab 8H4 labelled 61% and 85% of lymphocytes in the same organs. Mab 8H4 reacted with the majority of the lymphocytes in the thymus (98%). Mabs 1C2, 7A5 and 8H4 recognised surface markers on both Ig(-) and Ig(+) lymphocytes in peripheral blood and in spleen in double staining experiments. An increased proportion of Ig(-) lymphocytes were identified when Ig(+) lymphocytes were eliminated by immunomagnetic separation. No cross-reactivity of Mabs 1C2, 7A5 or 8H4 to anti-thrombocyte Mabs was detected. Mab 1C2 captured molecules of about 40 and also of 55-60kDa, in an immunoprecipitation assay. Mab 7A5 recognised an antigen of approximately 75-80kDa and Mab 8H4 identified proteins of about 70, 100 and 150kDa. Immunohistochemical staining by Mab 8H4 of fixed thymus, revealed a strong labelling of lymphoid cells in the outer zones of thymus. The 8H4 positive lymphoid cells surrounds circular structures, which were not labelled by Mab 8H4. These distinctly appearing structures have a similar shape as nurse cells described in mammals.     

Effect of sublethal concentrations of four chemicals on susceptibility of juvenile rainbow trout (Oncorhynchus mykiss) to saprolegniosis

The effects of sublethal concentrations of a variety of chemicals on the susceptibility of rainbow trout (Oncorhynchus mykiss) juveniles to Saprolegnia parasitica infection was examined. Sublethal concentrations of un-ionized ammonia (0.05 mg/liter) and nitrite (0.12 mg/liter) increased fish susceptibility after 10 days of exposure to the toxin, this increase being higher for ammonia (75% and 20% morbidity) than for nitrite (20% and 0% morbidity, respectively) with inoculum doses of 1.4 x 10(6) and 9.5 x 10(5) zoospores per liter, respectively. Sublethal concentrations of copper (0.05 mg/liter) or cyanide (0.05 mg/liter) did not show enhancement of infection by S. parasitica, even though the toxin exposure was for 21 days and the inoculum doses were higher than those for the experiments with the nitrogen compounds (4 x 10(6) and 3.2 x 10(6) zoospores per liter. However, infections began to appear in control animals.     

A link between ectoparasite infection and susceptibility to bacterial disease in rainbow trout

Rainbow trout, Oncorhynchus mykiss, were infected concomitantly with Argulus coregoni and Flavobacterium columnare and their survival was compared with that of fish infected with either the parasite or the bacterium alone. The mortality of fish challenged with A. coregoni was negligible while infection with F. columnare alone led to significantly lower survival. However, compared with single infections, the mortality was significantly higher and the onset of disease condition was earlier among fish, which were concomitantly infected by A. coregoni and F. columnare. This data presents, for the first time, experimental support for the hypothesis that an ectoparasite infection increases susceptibility of fish to a bacterial pathogen.