An outbreak of listeriosis suspected to have been caused by rainbow trout

An outbreak of listeriosis in Sweden, consisting of nine cases, was investigated by means of molecular typing of strains from patients and strains isolated from suspected foodstuffs, together with interviews of the patients. Listeria monocytogenes was isolated from six of the patients, and all isolates were of the same clonal type. This clonal type was also isolated from a "gravad" rainbow trout, made by producer Y, found in the refrigerator of one of the patients. Unopened packages obtained from producer Y were also found to contain the same clonal type of L. monocytogenes. Based on the interview results and the bacteriological typing, we suspect that at least six of the nine cases were caused by gravad or cold-smoked rainbow trout made by producer Y. To our knowledge, this is the first rainbow trout-borne outbreak of listeriosis ever reported.     

Purification and characterization of urotensin II from the brain of a teleost (trout, Oncorhynchus mykiss) and an elasmobranch (skate, Raja rhina)

Peptides related to urotensin II have been isolated in pure form from an extract of whole brain of a teleost, the rainbow trout (Oncorhynchus mykiss) and of an elasmobranch, the longnose skate (Raja rhina). The primary structure of the trout peptide [Gly-Gly-Asn-Ser-Glu-Cys-Phe-Trp-Lys-Tyr-Cys-Val] is similar to that of urotensin II peptides isolated from the urophyses of other teleost fish. For example, trout urotensin II contains only one amino acid substitution (Thr4-->Ser) compared with urotensin II beta 1 isolated from the urophysis of the carp. The primary structure of the skate peptide [Asn-Asn-Phe-Ser-Asp-Cys-Phe-Trp-Lys-Tyr-Cys-Val] is the same as urotensin II isolated from the caudal spinal cord region of the dogfish Scyliorhinus canicula. The data provide chemical evidence to support the conclusion of earlier morphological studies [Yulis, C. R., and Lederis, K. (1988) Gen. Comp. Endocrinol. 70, 301-311) that certain species of fish possess an extensive extraurophyseal distribution of urotensin II-immunoreactive neurons.     

Disruption of the blood-brain interface in neonatal rat neocortex induces a transient expression of metallothionein in reactive astrocytes

Exposure of the adult rat brain parenchyma to zinc induces an increase in the intracerebral expression of the metal-binding protein, metallothionein, which is normally confined to astrocytes, ependymal cells, choroid plexus epithelial cells, and brain endothelial cells. Metallothionein is expressed only in diminutive amounts in astrocytes of the neonatal rat brain, which could imply that neonatal rats are devoid of the capacity to detoxify free metals released from a brain wound. In order to examine the influence of a brain injury on the expression of metallothionein in the neonatal brain, PO rats were subjected to a localized freeze lesion of the neocortex of the right temporal cortex. This lesion results in a disrupted blood-brain interface, leading to extravasation of plasma proteins. From 16 h, reactive astrocytosis, defined as an increase in the number and size of cells expressing GFAP and vimentin, was observed surrounding the neocortical lesion site. Astrocytes and pial cells situated adjacent to the area of injury also became positively stained for metallothionein. At 3-6 days post-lesion, the highest level of reactive astrocytes expressing metallothionein was observed. Neo-Timm staining revealed that histochemically reactive zinc had disappeared from the lesion site. Extracellular albumin and metallothionein-positive astrocytes were absent approximately 2 weeks after the lesion, whereas reactive astrocytosis was still observed. These results show that a lesion of the neonatal rat brain induces a transient expression of metallothionein in reactive astrocytes, probably as a response to metals released from the site of the brain injury.     

cDNA cloning of the beta subunit of teleost thyrotropin

cDNA clones encoding the beta subunit of thyrotropin (thyroid-stimulating hormone; TSH) were isolated from a cDNA library made from the pituitaries of immature rainbow trout and sequenced. The precursor of rainbow trout TSH beta consists of 147 aa, which can be cleaved into a signal peptide (20 aa) and a mature protein (127 aa) containing one potential N-glycosylation site and 12 cysteine residues. The protein showed highest homology with human TSH beta (51%) and lesser homology with human follitropin (42%), human lutropin (32%), and salmon gonadotropin (31-33%) beta subunits. The identification of TSH in addition to two gonadotropins (gonadotropins I and II) in the teleost fish suggests that the divergence of three kinds of glycoprotein hormones from an ancestral molecule took place earlier than the time of divergence of teleosts from the main line of evolution leading to tetrapods. Northern blot analysis showed that the expression of the rainbow trout TSH beta gene is specific to the pituitary gland and is significantly higher in immature fish than in mature fish, suggesting that TSH plays some role in the biological processes of immature fish.     

Cloning of a cDNA coding for active tyrosine hydroxylase in the rainbow trout (Oncorhynchus mykiss): comparison with other hydroxylases and enzymatic expression

Although catecholamines are of critical importance for neuroendocrine function in teleost fishes, there has been no tool to give access to pretranslational regulation of their synthesis enzymes. In this study, we undertook cloning of the cDNA coding for the tyrosine hydroxylase (TH) of the rainbow trout (Oncorhynchus mykiss). First, we looked for a tissue sufficiently rich in TH to make an expression library. The cDNA coding for the rainbow trout TH (rtTH) was then cloned and sequenced. The rtTH sequence encodes a protein of 489 amino acids. Several domains and amino acids required for enzyme activity, like cysteines or phosphorylation sites, are highly conserved between species. Northern blot analysis showed a single rtTH messenger RNA of 4.2 kb expressed in the anteroventral brain. The ability of rtTH to hydroxylate L-tyrosine was analyzed by transient expression of the rtTH cDNA in COS-1 cells. In vitro TH activity, using COS-1 cell extracts, demonstrated that TH activity in transfected cells was 40-fold higher than in untransfected cells. Western blot analysis revealed a single protein of approximately 65 kDa in both COS-1 cells and in trout brain. This rtTH cDNA provides us with a tool for further studies on pretranslational regulation of the enzyme in salmonids.     

Evidence for an endogenous clock in the retina of rainbow trout: II. Circadian rhythmicity of serotonin metabolism

The purpose of the present study was to investigate patterns of circadian rhythmicity in the retina of a salmonid fish, the rainbow trout (Oncorhynchus mykiss). Our data present the first demonstration of intraretinal variations of serotonergic substances under light/dark-conditions (LD) and during continuous darkness (DD). All substances examined (serotonin, N-acetyl serotonin, 5-hydroxytryptophol, 5-hydroxyindole acetic acid) were rhythmic in LD. Serotonin, N-acetyl-serotonin, and 5-hydroxyindole acetic acid showed a preservation of specific features of rhythmicity in DD indicating the involvement of an endogenous pacemaker in the regulation of serotonin metabolism in the rainbow trout eye.     

Prokinetic drugs in digestive disorders

Adherence capacity to tissue substrate, endocytosis capacity for heterologous and homologous proteins, and proteolytic activity were determined in intestinal granulocytes (EGCs) isolated from healthy adult rainbow trout. The percentage of cells that could adhere to a smooth plastic surface increased with increasing incubation time. Endocytosis was effective for heterologous (human immunoglobulin G, IgGh; ovine somatotropin, oST) but not homologous proteins (recombinant trout somatotropin, rtST). The activity of cathepsin D increased significantly after the endocytosis of a heterologous protein. Finally, the analysis of immunoblots of homogenates of granulocytes incubated in the presence of the two different proteins was used to show the endocytosis and degradation of heterologous proteins. These results show that isolated EGCs can endocytose and degrade heterologous proteins.     

Cadmium toxicity and distribution in metallothionein-I and -II deficient transgenic mice

To date, numerous correlative studies have implicated metallothionein in the detoxification of heavy metals and in the regulation of metal distribution within an organism. In the present study cadmium-binding proteins (metallothionein equivalents), cadmium acute toxicity, and cadmium distribution in tissues and subcellular fractions were compared in metallothionein-I and -II deficient (MT-/-) mice and the parental strain carrying intact metallothionein genes (MT+/+) to determine if the absence of metallothionein altered any of these parameters. In an uninduced state, MT-/- mice expressed lower levels of cadmium-binding proteins relative to MT+/+ mice in several tissues. Administration of zinc enhanced the levels of cadmium-binding proteins in liver, small intestine, kidney, pancreas, and male sex organs, but not in cecum or brain of MT+/+ mice compared to zinc pretreated MT-/- mice. The cadmium LD50 was similar for MT-/-, MT+/+, and zinc-pretreated MT-/- mice (15-17 mumol CdCl2/kg body weight delivered i.p.). However, zinc-pretreated MT+/+ mice had a cadmium LD50 of 58-63 mumol CdCl2/kg body weight. Over two-thirds of cadmium was found in liver, cecum, small intestine, and kidney in both MT+/+ and MT-/- mice; therefore, metallothionein levels do not appear to play a major role in the tissue distribution of cadmium. However, after zinc pretreatment, MT+/+ mice accumulated more cadmium in the liver and less in other tissues, whereas the amount of cadmium in the liver was not altered by zinc pretreatment in MT-/- mice. In general, the cytosolic/particulate ratio of cadmium was significantly higher in tissues of noninduced MT+/+ mice relative to MT-/- mice. This difference was accentuated after zinc pretreatment. Together these results indicate that basal levels of metallothionein do not protect from the acute toxicity of a single i.p. cadmium challenge. Furthermore, it does not appear that the cytosolic compartmentalization of cadmium is correlated with reduced toxicity.     

Salmonid herpesvirus 2. Epizootiology and serological relationship

Herpesvirus infections of kokanee salmon, masu salmon, coho salmon, and rainbow trout have been reported in Japan. The 11 herpesvirus strains isolated from kokanee salmon (NeVTA), masu salmon (YTV and 3 strains of OMV), coho salmon (CSTV, COTV and 2 strains of OKV), rainbow trout (RKV and RHV), and Herpesvirus salmonis were compared for their serological relatedness by serum cross-neutralization tests with polyclonal rabbit antiserum. The herpesvirus strains isolated in Japan were neutralized by antisera against these viruses and were found to be closely related to salmonid herpesvirus 2 (OMV strain 00-7812). These strains were, however, clearly distinguished from salmonid herpesvirus 1 (H salmonis).     

A detailed linkage map of rainbow trout produced using doubled haploids

We report the first detailed genetic linkage map of rainbow trout (Oncorhynchus mykiss). The segregation analysis was performed using 76 doubled haploid rainbow trout produced by androgenesis from a hybrid between the "OSU" and "Arlee" androgenetically derived homozygous lines. Four hundred and seventy-six markers segregated into 31 major linkage groups and 11 small groups (< 5 markers/group). The minimum genome size is estimated to be 2627.5 cM in length. The sex-determining locus segregated to a distal position on one of the linkage groups. We analyzed the chromosomal distribution of three classes of markers: (1) amplified fragment length polymorphisms, (2) variable number of tandem repeats, and (3) markers obtained using probes homologous to the 5' or 3' end of salmonid-specific small interspersed nuclear elements. Many of the first class of markers were clustered in regions that appear to correspond to centromeres. The second class of markers were more telomeric in distribution, and the third class were intermediate. Tetrasomic inheritance, apparently related to the tetraploid ancestry of salmonid fishes, was detected at one simple sequence repeat locus and suggested by the presence of one extremely large linkage group that appeared to consist of two smaller groups linked at their tips. The double haploid rainbow trout lines and linkage map present a foundation for further genomic studies.     

Relative zinc-binding capacity of metallothionein: studies in renal cytosols from zinc-injected rats

Each rat was injected intraperitoneally once with 0.9% NaCl or zinc (10, 20, 40, or 60 mg zinc/kg b.w.). The zinc content in kidney reached a maximum level of approx. 50 microgram/g kidney, corresponding to a dose (40 mg zinc/kg b.w.). The distribution profiles of the renal cytosols of zinc-injected rats on a Sephadex G-75 column showed that most of the increased zinc was attributable to metallothionein. There was a linear relationship between the zinc contents in cytosol and metallothionein. Our results demonstrated that there was a limit of zinc accumulation in kidneys of zinc-injected rats and that 57% of the increased zinc in renal cytosols was bound to metallothionein. Our results suggest that the role of metallothionein in zinc accumulation in the kidney is similar to that of zinc accumulation in liver.     

Body surface mapping during pacing at multiple sites in the human atrium: P-wave morphology of ectopic right atrial activation

A magnetic cell sorting system has been optimised for the purification of rainbow trout neutrophils using a monoclonal antibody (E3D9) raised against Atlantic salmon neutrophils. The purified neutrophils have good viability (85%) and purity (approximately 92%), and were functional in respiratory burst and migration assays. The isolated neutrophils responded rapidly to PMA stimulation, producing levels of superoxide anion (4.85 nmols superoxide min-1/10(6) cells) approximately twice as high as macrophages from the same species. In the migration assay, there was a four-fold increase in migrating cells using the purified neutrophils compared with unfractionated blood leucocytes, and a relatively high neutrophil migratory activity was seen in the absence of serum.     

Repeated administration of alpha-hederin results in alterations in maternal zinc status and adverse developmental outcome in the rat

The administration of alpha-hederin, an inducer of metallothionein, results in a secondary zinc deficiency that may be an important maternally mediated mechanism of developmental toxicity. Previous studies have shown adverse developmental outcome with a single administration of alpha-hederin to rats on gestation day (GD) 8 or 11. The objective of this study was to determine whether dosing of alpha-hederin throughout organogenesis would result in a sustained elevation of maternal hepatic metallothionein and subsequent developmental abnormalities. Rats were administered dosage levels of 0 (vehicle only), 20, or 30 mumol/kg from GD 6-15. Maternal hepatic metallothionein levels were 10-fold higher on GD 16 in the treatment groups than the controls. Consequently, liver zinc concentrations increased 60% and 54%, whereas plasma levels decreased 23% and 33% in the 20 and 30 mumol/kg treatment groups, respectively. At GD 20, mean fetal weights of the treatment litters were 11% less than control litters. The administration of alpha-hederin resulted in a threefold increase in the number of offspring that exhibited developmental abnormalities, including visceral and skeletal malformations. Following an oral pulse of 65Zn subsequent to treatment with 0 or 20 mumol/kg of alpha-hederin, the distribution of 65Zn to the liver of treated dams was twice that of controls, whereas the radiolabeled zinc apportioned to the decidua and uterus decreased by 44%. Furthermore, the 65Zn detected in the embryos from treated dams was 70% lower than in embryos from control dams. In conclusion, low doses of a metallothionein inducer administered to the dam from GD 6-15 resulted in a sustained elevation of hepatic metallothionein and a subsequent redistribution of zinc leading to a decrease in the zinc available to the embryo and ultimately to adverse development of the offspring. Repeated dosing throughout organogenesis, as required in regulated safety assessment testing, increased the severity of the effects previously observed with single large dosages of the toxicant administered during midgestation.     

CYP4T1--a cytochrome P450 expressed in rainbow trout (Oncorhynchus mykiss) liver

Total RNA isolated from a rainbow trout (Oncorhynchus mykiss) liver was subjected to RT/PCR using degenerate primers designed from homologous regions amongst cytochrome P450 CYP4 proteins. PCR amplification resulted in a single electrophoretic band which was excised, purified and sequenced directly, using cycle sequencing. The deduced protein sequence demonstrated the closest amino acid identity to rabbit CYP4B1 (54.6%) and rat CYP4B2 (55.4%). Phylogenic analysis of this sequence was found to be significantly different to any other CYP4 sequence and has been named CYP4T1. This represents the first CYP4 family member to be identified in an aquatic vertebrate.     

Alx-4: cDNA cloning and characterization of a novel paired-type homeodomain protein

Fish react to handling and capture with a burst of exercise that affects them deeply. The present study examines the effect of such severe exercise and the time course of recovery on the hematology (including spleen response) and metabolism of a population of cultured rainbow trout. Exercise was induced by continuous chasing for 5 min when the trout showed signs of exhaustion. Such exercise led to spleen contraction and an increase in haematocrit values. Carbohydrates were mobilized and anaerobic glycolysis produced lactate without significant effect on lipid metabolism. The conclusion is reached that the respiratory properties of rainbow trout blood do not change following severe exercise, while muscle anaerobic metabolism is slightly activated as deduced from the fast and short lactacidemia observed, which may have been related to a reduced stressing component, as the exercise was performed in the same environment in which the fish were reared.     

Acute dietary PCPA treatment decreases the KD of brain [3H]5-HT binding in rainbow trout, Oncorhynchus mykiss, fed a high carbohydrate diet

Two experiments were conducted to determine the effect of dietary PCPA on brain [3H]5-HT binding in rainbow trout. [3H]5-HT binding was quantitatively similar to that observed in mammalian species. Dietary treatment with PCPA had no effect on the Bmax of [3H]5-HT binding in either experiment while the KD was decreased in Experiment 1. These results demonstrate that [3H]5-HT binding affinity is increased following short term inhibition of 5-HT synthesis by PCPA in rainbow trout.     

Measuring the unmeasurable: a caring science perspective on patient classification

The histone H3 (sH3) promoter of Atlantic salmon (Salmo salar) was cloned via polymerase chain reaction using primers designed from the rainbow trout (Oncorhynchus mykiss) promoter sequence. A comparison of the nucleotide sequence with the equivalent sequences from rainbow trout and sockeye salmon (Oncorhynchus nerka) revealed a high degree of conservation. In vivo expression analysis of the sH3 promoter was carried out in both rainbow trout and zebrafish (Danio rerio) embryos. A direct comparison of the sH3 promoter with the viral RSV promoter in rainbow trout resulted in stronger expression of the sH3 promoter. Furthermore, lacZ expression directed by the sH3 promoter was ubiquitous in several different cell types in developing zebrafish embryos. These results suggest that the sH3 promoter will be useful in transgenic studies in Atlantic salmon.