Real-time quantitative polymerase chain reaction (QPCR) to identify Myxobolus cerebralis in rainbow trout Oncorhynchus mykiss

This study describes the development of a TaqMan real-time quantitative polymerase chain reaction (QPCR) technique using the heat-shock protein 70 (Hsp 70) and 18S ribosomal DNA (18S rDNA) sequences to identify Myxobolus cerebralis and attempt to quantify infection severity within rainbow trout fry Oncorhynchus mykiss. Rainbow trout for this study were exposed to M. cerebralis under natural river conditions and examined for infection by histology, polymerase chain reaction (PCR) and QPCR analysis at 900 Celsius temperature units (CTUs) following exposure. Detection sensitivity by QPCR was shown to be equal to traditional PCR but greater than histopathology. Primer/probe combinations developed for this study were capable of specifically detecting M. cerebralis DNA in infected fish tissue and single triactinomyxon (TAM) spores with a sensitivity of 12.5 and 6.3 pg microl(-1) of DNA for the Hsp 70 and 18S rDNA sequences, respectively. A strong relationship between QPCR and infection severity was found for the Hsp 70 probe when parasite copy number and histology scores of 0-4 were compared (R2 = 0.96, p = 0.003). However, a reduction in copy number was observed at higher histology scores for the 18S probe (scores of 4 and 5) and the Hsp 70 probe (score of 5). The results of this study demonstrate that QPCR analysis is an effective tool for detecting M. cerebralis in fish tissue and may provide a relative indication of infection severity.     

Whirling disease: host specificity and interaction between the actinosporean stage of Myxobolus cerebralis and rainbow trout Oncorhynchus mykiss

Scanning electron microscopic studies were conducted on rainbow trout Oncorhynchus mykiss in the first 60 min after their exposure to the triactinomyxon spores of Myxobolus cerebralis. The results demonstrated that as early as 1 min post exposure the whole process, from the attachment of the triactinomyxon spores to the complete penetration of their sporoplasm germs, had occurred. The triactinomyxon spores sought out the secretory openings of mucous cells of the epidermis, the respiratory epithelium and the buccal cavity of trout and used them as portals of entry. Exposure experiments of the triactinomyxon spores of M. cerebralis to non-salmonid fish, such as goldfish Carassius auratus, carp Cyprinus carpio, nose Chondrostoma nasus, medaka Oryzias latipes, guppy Poecilia reticulata and also the amphibian tadpole Rana pipiens as well as to rainbow trout fry indicated a specificity for salmonids. Attempts to activate the triactinomyxon spores by exposure to mucus prepared from cyprinid and salmonid fish showed no significant differences from those conducted in tap water. The results suggest that the simultaneous presence of both mechano- and chemotactic stimuli was required for finding the salmonid fish host.     

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.     

Analysis of rainbow trout Oncorhynchus mykiss epidermal mucus and evaluation of semiochemical activity for polar filament discharge in Myxobolus cerebralis actinospores

As myxozoan actinospores are stimulated by fish epidermal mucus to attach to their hosts via extrusion of filaments from specialized organelles, the polar capsules, mucus components were tested for discharge triggering activity on Myxobolus cerebralis actinospores. Using various methodological approaches, a selective exclusion of candidate substances based on experimental outcome is provided and the physiochemical traits of the putative agents are explored to create a basis for the isolation of the host recognition chemostimuli. Activity was detected in compounds that can be characterized as small molecular, amphiphilic to slightly hydrophobic organic substances. They were separable by chromatographic methods using reversed phase C18 supports. An active fraction was isolated by solid phase extraction comprising at least nine UV-detectable constituents as shown by thin-layer chromatography. By means of biochemical fractionation and analysis of host fish mucus, non-volatile inorganic electrolytes, all volatiles, free L-amino acids, glycoproteins, bound and free hexoses, sialic acids, glycans, proteins, urea, amines and inositols were shown not to trigger polar filament discharge. The results contribute to the identification of the attachment host cues and enable a more focused laboratory activation of myxozoan actinospores.     

Studies on the infectivity of Diplostomum spathaceum in rainbow trout (Oncorhynchus mykiss)

The infectivity of Diplostomum spathaceum (Digenea: Trematoda) cercariae to rainbow trout and the efficacy of the diplostomule migration to the lens following different routes of administration was examined. The optimum age of infectivity for cercariae was between 0-5 h after liberation from the snail and for intraperitoneally injected diplostomules, 5 h post-transformation in vitro through fish skin. After exposure of the entire fish body or head to cercariae, metacercariae first appeared in the lens at 5 h and their numbers gradually increased until 22 h. Following exposure of the tail region of rainbow trout to cercariae, metacercariae first appeared in the lens at 14 h. Significantly more metacercariae established in the lens of fish following exposure of the fish head compared with the tail region; 40% of penetrating cercariae reached the lens of fish following exposure of the head or entire body, 20% of cercariae or diplostomules injected either intraperitoneally, intramuscularly or intracardially reached the lens while only 5% of cercariae established as metacercariae following exposure of the tail region.     

虹鳟对时间和地点的学习记忆(英文)

为确定虹鳟(Oncorhynchus mykiss)是否能将日投放两次食物的不同时间和地点联系起来,在30天内,于09:00-10:00将食饵投放在水族箱一侧,于15:00-16:00将食饵投放在水族箱另一侧.在第21天和28天,不放食饵以确定虹鳟对时间-地点的学习记忆.结果表明,虹鳟将不同时间和地点联系起来以获取食物,并且在水族箱两个投放食饵之处均表现出预取食活动,提示该物种具有控制时间-地点学习任务的内在机制.     

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.     

Cardiorespiratory responses to haemolytic anaemia in rainbow trout Oncorhynchus mykiss

To quantify cardiorespiratory response to experimental anaemia in rainbow trout Oncorhynchus mykiss, a 24 h phenylhydrazine treatment was used to reduce haematocrit to almost one third of its initial value over 4–5 days. In response, relative blood velocity in the ventral aorta (an index of cardiac output) progressively increased to more than double to its normocythaemic value and there was no significant change in routine oxygen uptake. Thus, the primary compensatory response to anaemia was an increase in cardiac output.     

Copper exposure and the degeneration of olfactory receptors in rainbow trout (Oncorhynchus mykiss)

Abstract

Sublethal concentrations of copper in water cause the degeneration of olfactory receptors in rainbow trout (Oncorhynchus mykiss). Receptor cell loss has been correlated to the loss of olfaction in fish and may cause difficulties in olfactory mediated behaviors such as migration. This study investigated the effects of three levels of copper (100, 75 and 50 mg L^?1) on the olfactory epithelium of rainbow trout. Twenty fish randomly allocated between three exposure groups and one control were exposed for 24 hours under static renewal conditions. Light and scanning electron microscopic observations of olfactory tissue were taken to determine the extent of degeneration of receptors. In addition, levels of copper and zinc in the brain tissues were analyzed to determine if the olfactory route was a significant route of copper exposure and transfer to fish brain tissue. Results indicate that degeneration of receptors is related to the concentration of copper. Levels of copper in brain were found to be below detection of the instrument. Levels of zinc were extremely variable ranging from 52 to 132 ng zinc g^?1 brain tissue.     

Response of serum carotenoid levels to dietary astaxanthin and canthaxanthin in immature rainbow trout Oncorhynchus mykiss

Rainbow trout were fed a diet supplemented with astaxanthin (89 mg/kg) or canthaxanthin (116 mg/kg) in two different experiments: experiment 1 was designed to measure the kinetics of the appearance and disappearance of carotenoids in the serum; experiment 2 was undertaken to establish the serum dose-response to synthetic astaxanthin and canthaxanthin for immature rainbow trout. The serum carotenoid concentrations of immature rainbow trout increased when fish were fed carotenoid supplemented feed and then reached a plateau after 1 day of intake for astaxanthin and after 2 days for canthaxanthin. Circulating astaxanthin represented a value 2.3 times that of canthaxanthin. After dietary supplementation was discontinued, the serum carotenoid concentrations decreased within 3 days for both carotenoids. The average decreasing slopes for the two carotenoid pigments were parallel, indicating a similarity in the rate of which astaxanthin and canthaxanthin are utilized by rainbow trout. The serum dose-response of trout that received dietary keto-carotenoids increased with increasing pigment levels. The hypothesis that absorption of dietary carotenoids in 12.5–200 mg/kg range of concentration across the gut wall may be by passive diffusion is proposed.     

Effects of exposure to sublethal propiconazole on intestine-related biochemical responses in rainbow trout,Oncorhynchus mykiss

The effect of long-term (30 days) exposure to PCZ (0.2, 50, and 500 μg l^?1) on intestine-related biochemical markers in rainbow trout was investigated. Multiple biomarkers were measured, including digestive enzymes (proteolytic enzymes and amylase), antioxidant responses (TBARS, CP, SOD, CAT, GR and GPx) and energy metabolic parameters (RNA/DNA ratio, Na⁺-K⁺-ATPase). Exposure to 500 μg l^?1 PCZ led to significantly inhibited (p < 0.01) proteolytic enzyme and amylase activity. Activities of the antioxidant enzymes SOD, CAT, and GPx gradually increased at lower PCZ concentrations (0.2 and 50 μg l^?1). At the highest concentration (500 μg l^?1), oxidative stress was apparent as significant higher (p < 0.05) lipid peroxidation and protein carbonyls, associated with an inhibition of antioxidant enzymes activity. Moreover, energy metabolic parameters (RNA/DNA ratio, Na⁺-K⁺-ATPase) were significantly inhibited (p < 0.01) in the intestines of fish exposed to 500 μg l^?1 PCZ, compared with controls. We suggest that long-term exposure to PCZ could result in several responses in intestine-related biochemical markers, which potentially could be used as indicators for monitoring residual PCZ present in the aquatic environment.     

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.     

Formation of chromosome aberrations in androgenetic rainbow trout Oncorhynchus mykiss

Residues of maternal nuclear DNA in the form of chromosome fragments were observed in the healthy and morphologically normal androgenetic rainbow trout Oncorhynchus mykiss. A hypothetical model for formation of chromosome re‐arrangements caused by the incomplete maternal nuclear DNA inactivation in the androgenetic rainbow trout was proposed in the present paper.     

Brain distribution of myosin Va in rainbow trout Oncorhynchus mykiss

This study presents data on myosin Va localization in the central nervous system of rainbow trout. We demonstrate, via immunoblots and immunocytochemistry, the expression of myosin Va in several neuronal populations of forebrain, midbrain, hindbrain and spinal cord. The neuronal populations that express myosin Va in trout constitute a very diverse group that do not seem to have many specific similarities such as neurotransmitters used, cellular size or length of their processes. The intensity of the immunoreactivity and the number of immunoreactive cells differ from region to region. Although there is a broad distribution of myosin Va, it is not present in all neuronal populations. This result is in agreement with a previous report, which indicated that myosin Va is approximately as abundant as conventional myosin II and kinesin, and it is broadly involved in neuronal motility events such as axoplasmatic transport. Furthermore, this distribution pattern is in accordance with what was shown in rats and mice; it indicates phylogenetic maintenance of the myosin Va main functions.     

Distribution of canthaxanthin in immature rainbow trout Oncorhynchus mykiss serum

• 1.1. The present study was undertaken in order to define the distribution of canthaxanthin between the lipoprotein fractions in serum of immature rainbow trout fed a diet supplemented with synthetic canthaxanthin (80 mg/kg).
• 2.2. Lipoproteins were separated by density-gradient ultracentrifugation.
• 3.3. Canthaxanthin was found in all lipoprotein fractions, in different amounts according to the density of the lipoprotein fraction: VLDL, 13.9%; LDL, 15.2% or LDL, 29.1% since the density of the first fraction was 1.006 g/ml; HDL, 60.4% and VHDL, 10.5%.

     

A blue variant in the rainbow trout, Oncorhynchus mykiss Walbaum

A blue variant of the rainbow trout, which appeared in a French fish farm, displayed an iridescent body color that was cobalt blue on the back, lighter on the undersides, and silvery on the belly and which held up to adult stage. This color was supposed to result from a Tyndall effect involving a structural arrangement of melanin pigments because it disappeared when it was associated with a depigmenting gene. This blue variant appeared to be governed by an autosomal recessive gene. Blue fry survival and body weight were about 25% less than those of wild-type sibs, but no major problem was observed in further breeding performances, including reproduction. These features do not correspond with those of the blue variants previously described in the rainbow trout.