Clinical and histopathological changes in rainbow trout Oncorhynchus mykiss experimentally infected with fungus Bradymyces oncorhynchi.

Bradymyces oncorhynchi is a poorly known melanised fungal species that has been isolated only from a hyperaemic focus near the enlarged spleen of a rainbow trout. Although the pathogenicity of this species to fish is suspected, it has not been fully confirmed. Four laboratory experiments were conducted to test the effect of the fungus on the health of rainbow trout fingerlings. Mycelia were cultivated under different conditions to increase inoculum variability and to test the impact of the conditions on the pathogenicity. The inoculum was subsequently administered by the intraperitoneal route. The clinical manifestations, gross pathological lesions and histopathological changes were identical in all experimental groups, i.e. lethargy, inappetence, anorexia, weight reduction, pale gills, full-thickness ulceration of the abdominal wall, muscle atrophy, haemorrhagic or pale liver, fat-altered pyloric region with haemorrhages, enlarged spleen, haemorrhagic ascites, systemic granulomatous lesions with the presence of melanised hyphae (phaeohyphomycosis), multifocal granulomatous hepatitis, perivascular lymphocytic infiltration, vacuolation of hepatocytes and vacuolation of the kidney tubule epithelium. No statistically significant differences were found in the survival of the fish with respect to experimental settings. The survival period ranged between 3 and 135 days.

Swim bladder mycosis in farmed rainbow trout Oncorhynchus mykiss caused by Phoma herbarum and experimental verification of pathogenicity.

In this study, spontaneous swim bladder mycosis was documented in a farmed fingerling rainbow trout from a raceway culture system. At necropsy, the gross lesions included a thickened swim bladder wall, and the posterior portion of the swim bladder was enlarged due to massive hyperplasia of muscle. A microscopic wet mount examination of the swim bladder contents revealed abundant septate hyphae, and histopathological examination showed periodic acid-Schiff-positive mycelia in the lumen and wall of the swim bladder. Histopathological examination of the thickened posterior swim bladder revealed muscle hyperplasia with expansion by inflammatory cells. The causative agent was identified as Phoma herbarum through morphological analysis and DNA sequencing. The disease was reproduced in rainbow trout fingerlings using intraperitoneal injection of a spore suspension. Necropsy in dead and moribund fish revealed extensive congestion and haemorrhages in the serosa of visceral organs and in liver and abdominal serosanguinous fluid. Histopathological examination showed severe hepatic congestion, sinusoidal dilatation, Kupffer cell reactivity, leukostasis and degenerative changes. Fungi were disseminated to the liver, pyloric caeca, kidney, spleen and heart. Although infections caused by Phoma spp. have been repeatedly reported in fish, species identification has been hampered by extensive taxonomic changes. The results of this study confirmed the pathogenicity of P. herbarum in salmonids by using a reliably identified strain during experimental fish infection and provides new knowledge regarding the course of infection.

Swim bladder mycosis in pretty tetra (Hemigrammus pulcher) caused by Exophiala pisciphila and Phaeophleospora hymenocallidicola, and experimental verification of pathogenicity.

Spontaneous invasive and chronic disseminated mycosis affected Hemigrammus pulcher kept in a public aquarium, and infection was manifested by inappetence, exophthalmia, erratic swimming, eroded scales, anaemia of the gills and abdominal distension. Internally, there was a grossly swollen swim bladder with a thickened wall filled with a dark mass. The body cavities contained a clear, light amber fluid and a swollen intestine which was full of a watery fluid containing small gas bubbles. Histopathology revealed a granulomatous inflammatory response with fungal hyphae in the lumen and wall of the swim bladder, hepatopancreas, spleen and kidneys with signs of nephrohydrosis. Exophiala pisciphila and Phaeophleospora hymenocallidicola were isolated from the swim bladder, abdominal cavity and gastrointestinal tract. The exogenous source of infection was probably the ample wooden decoration and plants inside the aquarium. Koch's postulates were fulfilled by re-isolation of both fungal species from fish artificially infected under laboratory conditions. As P. hymenocallidicola is less capable of defence against phagocytosis, E. pisciphila probably played a major role. Severe clinical manifestations with 100% mortality developed in two fish species infected by E. pisciphila. A significant increase in the plasma levels of amino acids was observed as a result of the activation of proteolysis.

Disseminated infection due to Exophiala pisciphila in Cardinal tetra, Paracheirodon axelrodi.

Cardinal tetra, Paracheirodon axelrodi (Schultz, 1956), kept in an ornamental tank, was found to be affected by severe invasive mycosis. Externally, the disease manifested as abdominal swelling, and internally, the anterior part of the intestine was extremely bloated with abundant dematiaceous septate hyphae and an accumulation of fluid. Histopathologically, a granulomatous inflammatory response was observed in the intestine wall, kidney and spleen. We assume that the mycotic agent was primarily deposited in the intestine and was then gradually disseminated to the other organs. DNA sequencing of ITS and LSU rDNA regions and phenotypic characterization were used for identification of the isolated fungus. The obtained data confirmed that the infection was caused by Exophiala pisciphila. The disease was subsequently reproduced in the carp fingerling using intramuscular and intraperitoneal injection of a spore suspension. The 13th day after intramuscular infection, a marked elevation of neutrophils was recorded in the peripheral blood; this involved a proliferation of band forms and segmented forms. As far as we know, this is the first report of infection due to E. pisciphila in Cardinal tetra.

Cephalotheca sulfurea (Ascomycota, Sordariomycetes), a new fungal pathogen of the farmed rainbow trout Oncorhynchus mykiss.

The first case of visceral mycotic infection due to Cephalotheca sulfurea (Cephalothecaceae, Ascomycota) is documented in farmed rainbow trout from a raceway culture system. The disease clinically manifested as a hyperaemic area in the liver of the fish, and histological examination using silver and PAS staining showed the presence of numerous foci of hyphae and spores. The causative agent was first isolated in pure culture from the liver and identified using morphological characteristics. Sequence data from ITS and LSU rDNA also clearly confirmed C. sulfurea as the causal agent. The pathogenicity of related species belonging to the family Cephalothecaceae has been well-documented in humans and dogs (superficial as well as systemic infections). However, C. sulfurea has never been reported as a pathogen of humans or animals, including marine and freshwater fishes. The morphological identification of C. sulfurea is difficult due to its similarity to several different fungal genera, and molecular methods are strongly recommended for reliable identification.

Effect of polychlorinated biphenyls (Delor 103) on haematological and enzyme parameters of the rainbow trout Oncorhynchus mykiss.

Rainbow trout at a weight of 223+/-12 g (mean+/-SD) were experimentally injected with a technical mixture of Delor 103 to evaluate the red blood cell indices (red blood cell count, haematocrit, haemoglobin, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration) and some biochemical and enzyme parameters of the blood plasma (total protein, glucose, inorganic phosphate, total calcium, sodium, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, lactate dehydrogenase). Delor 103, administered by the i.p. route at a concentration of 0.24 g kg(-1) 120 h(-1), caused an increase in the red blood cell counts, haematocrit values, haemoglobin concentrations, inorganic phosphate, alanine aminotransferase and lactate dehydrogenase. The sodium level fell. The fish injected with Delor 103 showed a relative decrease in the lymphocyte count and a relative increase in the count of neutrophile band forms.

Haematological analyses in rainbow trout Oncorhynchus mykiss affected by viral haemorrhagic septicaemia (VHS).

Rainbow trout Oncorhynchus mykiss weighing 87 +/- 15 g (mean +/- SD) were infected with viral haemorrhagic septicaemia virus (VHSV) and the haematological and biochemical profiles of peripheral blood examined. Depending on the clinical signs and gross pathology, the fish were divided into 2 groups: Group A included fish in the acute stage, Group B comprised fish in the chronic stage. Red blood cells were subjected to 6 haematological tests and blood plasma to 14 biochemical tests, which provided findings on changed substrate concentrations and enzyme activities. Diseased fish, compared to healthy fish, had a significantly lower red blood cell count, and lower haematocrit and haemoglobin levels. As for the biochemical parameters, the fish had less total protein, creatinine, glucose, triacylglycerol, inorganic phosphate, total calcium and sodium, and more blood urea, nitrogen and potassium. Uric acid levels remained unchanged. Increases were recorded in the catalytic concentration of alanine aminotransferase, aspartate aminotransferase and lactate dehydrogenase. A decrease was recorded in the catalytic concentration of alkaline phosphatase. Fish with VHS in the chronic stage, compared with healthy fish, were in worse condition, with a significantly reduced Fulton coefficient and Clark coefficient, and a higher hepatosomatic index and visceral somatic index.

Effect of repeated stress and administration of phenobarbital on the lymphoid tissue and on protein metabolism in the lymphocytes of infant and adult rats.

Further study of the response to chronic stress stimulation in the early postnatal phase showed that the i.p. injection of physiological saline (stress stimulation) induced lymphopenia, a 50% decrease in the incorporation of 3H-leucine into isolated lymphocytes and a decrease in the weight of the thymus in 7-day-old male rats. No such changes were observed in adult animals. If repeated doses of phenobarbital were administered to stressed young rats, however, lymphopenia did not occur and the rate of the incorporation of 3H-leucine into isolated lymphocytes was not different from the control value; the protein content of the lymphocytes was significantly raised, however. In adult animals, phenobarbital increased the rate of incorporation of 3H-leucine into the lymphocytes. The repeated administration of phenobarbital reduced the weight of the thymus in both young and adult animals, but a decrease in spleen weight was recorded only in the young animals. A single i.p. injection of ACTH or dexamethasone caused lymphopenia and slowed down the incorporation of 3H-leucine into the lymphocytes of both young and adult animals. The results show that the striking decrease observed in the rate of the liver metabolism of corticosterone in suckling young rats not injured by repeated stress stimulation is accompanied by significant changes in the lymphoid tissue.

Capillostrongyloides Ancistri sp. n. (Nematoda: Capillariidae) a new pathogenic parasite of aquarium fishes in Europe.

A new nematode species, Capillostrongyloides ancistri sp. n., is described from the intestine of aquarium-reared catfish Ancistrus dolichopterus Kner in Czechoslovakia. This fish is of South American origin and, therefore, C. ancistri sp. n. has probably been brought into aquaria in Europe from there. The parasite is characterized mainly by the structure of the male caudal end (presence of two large postanal papillae and the cuticular bursa supported by two caudal lobes), the length of a spicule (0.258-0.297 mm), the presence of a non-spiny spicular sheath, shape of the female end, structure of the stichosome (23-30 stichocytes present), and the size of the eggs. The species appears to be highly pathogenic to aquarium-reared Ancistrus dolichopterus, causing death of these fish.

Regulation of corticosterone metabolism in liver cell fractions in young and adult rats: cofactor requirements, effects of stress and phenobarbital treatment.

Corticosterone metabolism was studied in the 10,000 X g supernatant fraction of the liver homogenate supplemented with cofactors (NADH, NADPH), or with the system participating in NADPH synthesis. NADPH was more effective than NADH for the degradation of the A ring and the side chain of corticosterone. The rate of reduction of the A ring, in both the supernatant and the sediment, was higher in adult than in infant rats. The rate of metabolism of the side chain did not change during development in the supernatant, but it was lower in the sediment from adult than from young animals. Corticosterone metabolism was also studied in infant and adult rats, after recurrent stressful stimulation or the repeated administration of phenobarbital, in both liver homogenate fractions, supplemented by the NADPH-regenerating system. Both stress and phenobarbital administration reduced the rate of corticosterone side chain and A ring metabolism in the liver of 7-day-old young. In adult animals, the rate of corticosterone metabolism was unaffected by stress, but the administration of phenobarbital raised the rate of metabolism of the corticosterone side chain in the sediment fraction obtained by centrifugation at 10,000 X g.

Regulation of liver corticoid metabolism during early postnatal life: effects of surgical stress.

Corticosterone metabolism by liver slices was investigated in suckling (10-day-old), weanling (21-day-old) and adult male rats. During the suckling period adrenalectomy as well as sham adrenalectomy increase the rate of steroid A ring reduction and also the rate of steroid side chain degradation by 20-40%. In older animals such changes were not detected. The results support an earlier assumption that liver steroid metabolism is regulated in an age specific manner.

Properties of mitochondrial adenosine triphosphatase isolated from rat liver.

Soluble, oligomysin-insensitive ATPase was isolated from liver mitochondria by a new technique [Drahota and Houstek 1977]. Study of the resultant enzyme preparation provided further evidence that the isolated protein displays properties typical of mitochondrial ATPase (specific activity about 100 U/mg, optimum pH 8.2, activation by various bivalent cations and reassociation with membranes deprived of ATPase).

Corticosterone synthesis and serum levels at the end of the perinatal period a study of the effects of stress, diazepam and polyethylene glycol treatments.

The effects of stress (intraperitoneal injections onece daily for three days), diazepam (Faustan, Germed) and polyethylene glycol (Macrogolum, Spofa) on the corticosterone production rate (BPR) and concurrent changes in serum corticosterone levels (BSL) were investigated in 4-day-old male rats. Stress stimulation increased the BSL but the BPR was affected only slightly and the difference was not statistically significant. A low dose of diazepam (1 mg/kg) prevented the stress-induced rise in BSL and decrease of BPR, in comparison to stressed animals. On the contrary, high doses of diazepam (10 mg/kg) increased the BSL, while BPR corresponded to control values. Polyethylene glycol (0.12 g/kg; substance of this class is contained in the diazepam vehiculum) decreased the BSL, but the BPR was increased above control values; a higher dose of this substance (1.2 g/kg) yielded only scattered, non-significant results. It is concluded that in preweaned animals 1. the changes in BSL may be associated with minor or transient changes in BPR which indicate small changes in brainpituitary adrenocorticotropic activity, 2. changes in peripheral corticosterone metabolism can play an exceptionally important role in the regulation of glucocorticoid activity in very young rats, 3. a comparison of controls with stressed animals which had received low doses of diazepam indicate a relatively high steady state activity of adrenal cortex and its regulation in 4-day-old control rats.