Anorexia reduces the severity of cryptobiosis in Oncorhynchus mykiss

Cryptobia salmositica multiplied more rapidly and caused a more severe disease with a higher mortality (65%) in rainbow trout (Oncorhynchus mykiss) fed a 52% protein diet than in fish fed 37 or 22% protein diets (25 and 30% mortalities, respectively). Also, plasma protein was significantly higher in fish fed a 52% protein diet than in fish fed 37 or 22% protein diets. Anorexia in infected trout was related positively to parasitemia and was most significant at 4 wk postinfection. During the chronic phase of the infection, food consumption increased with declining parasitemia. It is hypothesized that anorexia lowers plasma protein level, which reduces the multiplication rate of parasites, thus decreasing the severity of the disease.     

In vitro attenuation of Cryptobia salmositica and its use as a live vaccine against cryptobiosis in Oncorhynchus mykiss

The present communication reports on the attenuation of a pathogenic hemoflagellate, Cryptobia salmositica Katz (Sarcomastigophora: Kinetoplastida) and its use as a live vaccine against cryptobiosis. The parasite was attenuated by continuous in vitro culture (at 10 C for 55 wk) in minimum essential medium. Attenuated (culture) forms are morphologically similar to virulent (blood) forms. They are however more slender and have a shorter anterior flagellum and a smaller nucleus and kinetoplast. The attenuated form returned to its normal size and multiplied when inoculated into naive Oncorhynchus mykiss. It produced a low parasitemia but did not cause disease (e.g., no exophthalmia or anemia) in fish. At four wk after infection, the vaccinated fish were challenged with the virulent parasite. They were protected from the disease, whereas the control (naive) fish, infected with only the virulent parasite, had the usual clinical signs (e.g., anemia, exophthalmia). No parasite was detected in any of 10 vaccinated fish at 22 wk after challenge with the virulent parasite. However, 5 of 9 fish infected with culture forms and 6 of 9 fish infected with blood forms still had detectable parasites at 26 and 22 wk after infection, respectively.     

The in vitro effects of isometamidium chloride (Samorin) on the piscine hemoflagellate Cryptobia salmositica (Kinetoplastida, Bodonina)

Isometamidium chloride (Samorin) is therapeutic in rainbow trout (Oncorhynchus mykiss) during preclinical and chronic cryptobiosis. However, the toxic mechanism of isometamidium on Cryptobia salmositica has not been elucidated. The objective of the present study was to examine the in vitro effects of isometamidium on C. salmositica. Under in vitro conditions, isometamidium chloride reduced the infectivity of C. salmositica suspended in whole fish blood. It accumulated rapidly in the kinetoplast (within 1 min) and caused disruption and decantenation of kinetoplast DNA. The in vitro cryptobiacidal activity of isometamidium was reduced when parasites were incubated in medium containing serum supplement, suggesting that isometamidium also binds to plasma proteins. Isometamidium altered glycoprotein receptors (epitopes) for antibodies on the surface of C. salmositica and thus protected some of the parasites from lysis by complement-fixing antibodies. In vitro oxygen consumption and carbon dioxide production decreased in drug-exposed C. salmositica, with increased products of glycolysis, i.e., lactate and pyruvate, after exposure to isometamidium. This suggests that some C. salmositica switched from aerobic respiration to glycolysis when the mitochondrion was damaged by isometamidium.     

In vitro effects of fetal bovine serum and glucose on multiplication of Cryptobia salmositica

Cryptobia salmositica multiplied rapidly at 10 C in a minimum essential medium (containing 1.0 mg glucose/ml, Hanks' salts and L-glutamine) supplemented with heat-inactivated fetal bovine serum (FBS) and HEPES buffer (25 mM). The multiplication rate of C. salmositica was related to the amount of FBS; the peak number (approximately 9 x 10(6) parasites/ml) was attained in about 6 wk when the medium contained 25% final concentration of FBS. Glucose utilization was related to the number of parasites; the maximum utilization was reached before peak numbers. Formation of rosette colonies was correlated with multiplication rate and numbers of parasites in cultures. Degenerate round forms found in old cultures probably were caused by the accumulation of metabolic wastes in the medium.     

Cryopreservation of Plasmodium chabaudi. II. Cooling and warming rates

Various cooling and warming rates were investigated to determine the optimum conditions for cryopreserving the intraerythrocytic stages of Plasmodium chabaudi. Infected blood, equilibrated in 10% v/v glycerol at 37 degrees C or in 15% v/v Me2SO at 0 degree C for 10 min, was cryopreserved using cooling rates between 1 and 5100 degrees C min-1. After overnight storage in liquid nitrogen the samples were warmed at 12,000 degrees C min-1. Warming rates between 1 and 12,000 degrees C min-1 were investigated using samples previously cooled at 3600 degrees C min-1. After thawing, the glycerol and Me2SO were removed by dilution in 15% v/v glucose-supplemented phosphate-buffered saline. Survival was assayed by inoculation of groups of five mice each with 10(6) infected cells and the time taken to reach a level of 2% parasitemia estimated. The optimum cooling rate was 3600 degrees C min-1 for parasites frozen using either 10% glycerol or 15% Me2SO; the pre-2% patent periods were 0.90 and 1.01 days above control values (representing survival levels of 21 and 17.5%, respectively). The optimum warming rate was 12,000 degrees C min-1; the pre-2% patent periods were 1.01 and 1.32 days above control values, respectively (18 and 10% survival), for glycerol and Me2SO. With ethanediol (5% v/v) and sucrose (15% w/v) as cryoprotectants the optimum warming rates were also 12,000 degrees C min-1 while the optimum cooling rates were 330 and 3600 degrees C min-1, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Second-line treatment for Helicobacter pylori infection: 10-day moxifloxacin-based triple therapy versus 2-week quadruple therapy

BACKGROUND AND AIM: The aim of this study was to test the efficacy of 10-day moxifloxacin-based triple therapy versus 2-week quadruple therapy for the second-line treatment of Helicobacter pylori infection. METHODS: One hundred and ninety-two patients who had failed previous H. pylori eradication on standard triple therapy were randomized to one of two regimens: 1, moxifloxacin (400 mg q.d.), amoxicillin (1000 mg b.i.d.), and esomeprazole (20 mg b.i.d.) for 10 days (the 10MEA group), or 2, esomeprazole (20 mg b.i.d.), tripotassium dicitrate bismuthate (300 mg q.i.d.), metronidazole (500 mg t.i.d.), and tetracycline 500 mg (q.i.d.) for 14 days (the 14EBMT group). The eradication rates, drug compliances, and side-effect rates of these two regimens were compared. RESULTS: Eradication rates by intention-to-treat and per-protocol analyses in the 10MEA and 14EMBT groups were 71.9% and 82.6%, and 71.7% and 90.5% (p = .973 and .321), respectively. The 10MEA group was significantly superior to the 14EMBT group in terms of side-effect rates (12.2% vs. 39.6%, p = .001), and discontinuation rates due to side-effects were lower in the 10MEA group than in the 14EMBT group (0.7% vs. 13.2%, p < .001). Moreover, compliance was higher in the 10MEA group (94.2% (131/139)) than in the 14EBMT group (83.0% (44/53)) (p = .014). CONCLUSION: The 10-day moxifloxacin-based triple therapy was found to have a high eradication rate with few side-effects and good drug compliance. These findings suggest that this regimen is a safe and effective second-line treatment option for H. pylori infection in Korea.     

A bloodstream Trypanosoma congolense sialidase could be involved in anemia during experimental trypanosomiasis

The release of Sialic acid (SA) into the serum by Trypanosoma congolense infected BalbC mice was investigated. A progressive increase in the level of serum SA corresponding to anemia and parasitemia was observed. At maximum parasitemia, the level of total SA from the red blood cells (RBC) dropped by about 45%. Solved polynomials revealed an association between free serum SA and RBC-SA. Positive roots of quadratics were used to predict complete cleavage of RBC-SA on day 7.01 and maximum accumulation of free serum SA on day 6.6. A steady rise in the level of serum sialidase (SD) activity and a low packed cell volume (PCV) with an increase in parasitemia were observed. Mice infused with galactose, methyl-beta-gal, lactose, mannose, or L-arabinose and challenged by intraperitoneal inoculation with Trypanosoma congolense neither developed anemia nor secreted free SA above the control level even though there was detectable SD activity. Bloodstream Trypanosoma congolense parasites were isolated using DEAE cellulose from heparinized blood of experimentally infected BalbC mice. The parasites were lysed with 0.2% Triton-CF 54 to release membrane bound SD. The activity of the SD was proportional to the number of parasites. The enzyme was partially purified on Q-Sepharose and Fetuin agarose columns successively. The final active fraction from the latter column was used as the partially purified SD. The enzyme had an optimum pH of 6 and was maximally active at 37 degrees C with a requirement for the divalent ions Ca(2+) and Mg(2+). The enzyme was highly specific for NeuAc5alpha2,3 lac and Methylumbelliferyl-Neu5Ac (4-MU-Neu5Ac) with K(M) values of 0.34 and 0.025 mM, respectively. It was inhibited competitively by 2,3-didehydroneuraminic acid (Neu5Ac2en) and para-nitro-phenyloxamic acid (pNPO) with inhibition binding constants K(i) of 65 and 215 micro M, respectively. In deviation from the procyclic trypanosomal SD, it lacked trans-sialidase (TS) activity. The possible role of a secreted bloodstream Trypanosoma congolense SD and the development of anemia in the pathogensesis of trypanosomiasis are discussed.     

Is fluid-phase endocytosis conserved in hepatocytes of species acclimated and adapted to different temperatures?

Our primary objective was to determine if rates of fluid-phase endocytosis (FPE) were conserved in hepatocytes from organisms acclimated and adapted to different temperatures. To this aim, the fluorescent dye Lucifer yellow was employed to measure FPE at different assay temperatures (AT) in hepatocytes from 5 degrees C- and 20 degrees C-acclimated trout, Oncorhynchus mykiss (at 5 and 20 degrees C AT), 22 degrees C- and 35 degrees C-acclimated tilapia, Oreochromis nilotica (at 22 and 35 degrees C AT), and the Sprague-Dawley rat (at 10, 20, and 37 degrees C AT). FPE was also studied in rats fed a long-chain polyunsaturated fatty acid (PUFA)-enriched diet (at 10 degrees C AT). Despite being temperature dependent, endocytic rates (values in pl. cell(-1). h(-1)) in both species of fish were compensated after a period of acclimation. For example, in 20 degrees C-acclimated trout, the rate of endocytosis declined from 1.84 to 1.07 when the AT was reduced from 20 to 5 degrees C; however, after a period of acclimation at 5 degrees C, the rate (at 5 degrees C AT) was largely restored (1.80) and almost perfectly compensated (95%). In tilapia, endocytic rates were also temperature compensated, although only partially (36%). Relatively similar rates obtained at 5 degrees C in 5 degrees C-acclimated trout (1.8), at 20 degrees C in 20 degrees C-acclimated trout (1.84), and at 22 degrees C in 22 degrees C-acclimated tilapia (2.2) suggest that endocytic rates are somewhat conserved in these two species of fish. In contrast, the rate in rat measured at 37 degrees C (16.83) was severalfold greater than in fish at their respective body temperatures. A role for lipids in determining rates of endocytosis was supported by data obtained at 10 degrees C in hepatocytes isolated from rats fed a long-chain PUFA-enriched diet: endocytic rates were higher (5.35 pl. cell(-1). h(-1)) than those of rats fed a standard chow diet (2.33 pl. cell(-1). h(-1)). The conservation of endocytic rates in fish may be related to their ability to conserve other membrane characteristics (i.e., order or phase behavior) by restructuring their membrane lipid composition or by modulating the activities of proteins that regulate endocytosis and membrane traffic, whereas the lack of conservation between fish and rat may be due to differences in metabolic rate.     

Temperature dependent activation of leucocyte populations of rainbow trout, Oncorhynchus mykiss, after intraperitoneal immunisation with Aeromonas salmonicida

The temperature dependence of in vivo activation of rainbow trout Oncorhynchus mykiss, leucocyte populations after intraperitoneal injection (i.p.) of fish with a T-cell independent antigen Aeromonas salmonicida (strain MT423) was investigated using a proliferation assay and flow cytometric analysis with mab specific for trout leucocyte surface markers. In trout kept at 15-17 degrees C a prominent activation of blood and spleen leucocytes was found. Also, drastic changes of the percentage of the leucocyte populations in blood and spleen occurred: the amount of monocytes in the blood increased between day 2 and day 7 post injection (p.i.), whereas in spleen the amount of monocytes stayed at a high level (approximately 35%) after a depression between day 4 and day 7 p.i. The percentage of B-lymphocytes was increased first in spleen and then in blood. The percentage of granulocytes in blood was elevated during the whole experiment compared to control fish. In trout kept at 10-12 degrees C only blood leucocytes showed a weak activation after i.p. injection of A. salmonicida, whereas spleen leucocytes showed nearly no reaction. Only the percentage of granulocytes in the blood (day 2-14 p.i.) and of monocytes in the spleen (day 2 and day 8 p.i.) was changed compared to phosphate buffered saline (PBS)-injected fish. However, the development of A. salmonicida specific antibodies was contrary to the cellular reaction. Whereas antibodies could first be detected after 16-18 days p.i. in both groups the amount of antibodies was significantly higher in sera of trout kept at 10-12 degrees C at day 22 and day 28 p.i. than in sera of trout kept at 15-17 degrees C. These results indicate stronger A. salmonicida induced activation of monocytes, granulocytes and B-lymphocytes at higher temperature. However, the development of a specific antibody response against A. salmonicida seemed to be more effective at lower temperatures.     

In vitro oxygen consumption and motility of Cryptobia salmositica, Cryptobia bullocki, and Cryptobia catostomi (Sarcomastigophora: Kinetoplastida).

Cryptobia salmositica (pathogenic and vaccine strains), Cryptobia bullocki (pathogenic), and Cryptobia catostomi (nonpathogenic) have similar oxygen consumption rates (0.17 +/- 0.01 nm O2/10(6) parasites). Incubation with sodium azide (5 microliters of a 1-M solution to 1 ml of parasite suspension, i.e., a 5-mM final concentration) reduced the oxygen consumption by approximately 4.5-fold. Motility of the parasites was also greatly reduced in sodium azide. The oxygen consumption and motility of the parasites returned to preazide treatment levels when the azide was removed even after 24 hr of incubation in sodium azide. The activities of hexokinase, pyruvate kinase, and cytochrome C oxidase were not detected in the 3 species of Cryptobia.     

Protection against Cryptobia (Trypanoplasma) salmositica and Salmonid Cryptobiosis

Cryptobia (Trypanoplasma) salmositica is a haemoflagellate that causes morbidity and mortality in salmon, Oncorhynchus spp, on the Pacific coast of North America. In this review, Patrick Woo briefly describes the pathogen, its transmissions (either indirectly via its leech vector, Piscicola salmositica, or directly between fish) and the clinical signs of the disease. He then outlines strategies that have been developed to protect fish against the pathogen, and the mechanism of innate resistance to disease in Cryptobia-tolerant fish. Protective strategies include the breeding of fish that are resistant to infection, and the use of an attenuated C. salmositica strain to protect susceptible fish from disease for at least two years. He ends the review with suggestions for further research that include the use of the leech vector to deliver the vaccine and the development of more novel protective strategies (eg. immunochemotherapy, anti-idiotype vaccine) against cryptobiosis.     

Cryptobiosis and its control in North American fishes

Cryptobiosis is caused by the haemoflagellates Cryptobia bullocki and Cryptobia salmositica. These parasites infect food fishes (e.g. flounders, salmon) on both the Atlantic and Pacific coasts of North America and clinical signs of the disease include anaemia, and abdominal distention with ascites. The virulent factor in salmonid cryptobiosis, caused by C. salmositica, is a secretory metalloprotease (200 kDa). Fish mortality may be up to 100% in the absence of treatment, consequently strategies have been developed to protect them from disease/mortality. A single dose of a live vaccine protects fish for at least 2 years, and it is via the production of complement-fixing antibodies, enhanced phagocytosis and cell-mediated cytotoxicity. Inhibition of the parasite's cysteine protease by a monoclonal antibody reduces multiplication, infectivity and survival of the parasite. Consequently, the recombinant cysteine protease (49 kDa) of the parasite will be tested as a potential vaccine. The trypanocidal drug, isometamidium chloride (1.0 mg/kg), is effective (therapeutic and prophylactic) against C. salmositica in chinook salmon. Its efficacy is significantly enhanced if it is conjugated either to a monoclonal antibody or to polyclonal antibodies from immune fish. Selective breeding of Cryptobia-resistant brook charr (innate resistance to infection) is possible, and the resistant factor(s) is controlled by a dominant Mendelian locus. In these resistant charr the parasite is lysed via the alternate pathway of complement activation (innate immunity to infection). There are also Cryptobia-tolerant charr, fish that are susceptible to infection but have no clinical disease (innate resistance to disease). In these fish, one of the natural anti-proteases, alpha2-macroglobulin, neutralises the metalloprotease secreted by C. salmositica. Production of transgenic Cryptobia-tolerant salmon is an option to vaccination and or chemotherapy. Also, transgenic pathogen-tolerant animals may be an alternate strategy against other pathogens where the disease mechanism is similar to cryptobiosis.     

Factors affecting the sporulation capacity during long-term storage of the aphid-pathogenic fungus Pandora neoaphidis grown on broomcorn millet

Aphid-pathogenic fungus, Pandora neoaphidis, grown on broomcorn millet possesses greater sporulation capacity (C(s)) than aphid cadavers. The most sporulating cultures (32.0x10(4) spores millet(-1) grain) with water content (C(w)) of 48.7% were prepared by incubation at 20 degrees C for 15 days and used to study the effect of temperature and humidity on C(s) during long-term storage. Cultures were sealed with paper to retain ambient humidity, with parafilm for saturated humidity, or kept in 85% and 98% RH chambers. The C(w) and C(s) were monitored during 200-day storage at 5-20 degrees C. The paper-sealed cultures at 5 degrees C, associated with 21-25% of C(w), were best preserved and their 120-day C(s) was similar to that of the fresh cadavers. Consistently or variably high RH at 5 degrees C resulted in significantly higher C(w) and lower C(s) despite longer viability. The regimes at 10 degrees C preserved the cultures for 40 days. The observations fit well to the logistic model C(s)=35.28/{1+exp[-2.36+(-0.003C(w)+0.001C(w)T)t]} (r(2)=0.95) for all regimes of temperature (T) or C(s)=35.55/[1+exp(-2.33+0.001C(w)t)] (r(2)=0.93) at 5 degrees C only. The rate of decline of C(s) of -0.003C(w)+0.001C(w)T or 0.001 C(w) over days (t) highlights the primary effect of C(w). The daily C(s)-decline rates obtained for the best-stored cultures and air-dried cadavers stored at 5 degrees C were surprisingly identical. The results suggest a possible cheap method for preparing and storing large quantities of P. neoaphiodis inocula.     

Novel horizontal transmission route for Enteromyxum leei (Myxozoa) by anal intubation of gilthead sea bream Sparus aurata

The aim of the present study was to determine whether Enteromyxum leei, one of the most threatening parasitic diseases in Mediterranean fish culture, could be transmitted by peranal intubation in gilthead sea bream Sparus aurata L. Fish were inoculated either orally or anally with intestinal scrapings of infected fish in 3 trials. Oral transmission failed, but the parasite was efficiently and quickly transmitted peranally. Prevalence of infection was 100% at 60 d post inoculation (p.i.) in Trial 1 under high summer temperature (22 to 25 degrees C; fish weight = 187.1 g), and 85.7% in just 15 d p.i. in Trial 3 using smaller fish (127.5 g) at autumn temperature (19 to 22 degrees C). In Trial 2, prevalence reached 60% at 60 d p.i. in the group reared at constant temperature (18 degrees C), whereas no fish was infected in the group that was kept at low winter temperature (11 to 12 degrees C), although infection appeared (46.1% at 216 d p.i.) when temperature increased in spring. The arrested development at low temperature has important epidemiological consequences, as fish giving false negative results in winter can act as reservoirs of the parasite. Histopathological examination showed a posterior-anterior intestinal gradient in the progression of the infection, in terms of both intensity and parasite maturation. Thus, per-anal intubation provides a very uniform, reliable and faster mode of transmission of E. leei than the commonly used transmission methods (cohabitation, exposure to infected effluent and oral inoculation), which require long exposure times or give variable and unpredictable results.     

Susceptibility of juvenile sole Solea senegalensis to marine isolates of viral haemorrhagic septicaemia virus from wild and farmed fish

The susceptibility of sole Solea senegalensis to infection with 3 viral haemorrhagic septicaemia virus (VHSV) strains obtained from wild Greenland halibut Reinhardtius hippoglossoides and farmed turbot Psetta maxima was demonstrated. Fish were infected by an intraperitoneal (i.p.), immersion or cohabitational route, and maintained at 16 degrees C. Infection trials showed that VHSV isolates were pathogenic for sole fingerlings by i.p. injection and waterborne exposure causing moderate levels of mortality (10 to 55%). In addition, the mortality observed in fish cohabitating with i.p.-infected sole confirms horizontal transmission of the virus. However, the low rates of mortality registered in this challenge suggest that there is a low dissemination of virus by the i.p.-infected sole, which results in lower secondary challenge of the cohabitating fish. External signs of disease included haemorrhaging of the ventral area and ascitic fluid in the body cavity. Dead fish were tested for VHSV by both cell culture and RT-PCR assay, using pools of kidney and spleen from 10 individuals. Virus was recovered from most of the pools composed of dead fish. The results obtained in this study not only demonstrate the susceptibility of sole to the VHSV strains employed but also indicate that wild VHSV marine isolates represent a potential risk for sole aquaculture.     

Trypanosoma evansi: Activities of adenine nucleotide degradation enzymes in cerebral cortex of infected rats

This study aimed to evaluate the activities of the ectoenzymes NTPDase and 5′-nucleotidase in synaptosomes from cerebral cortex of rats experimentally infected with Trypanosoma evansi. The animals were divided in four groups (n = 10) according to the time and degree of parasitemia (groups A, B, C and D). The animals from group A were euthanized on day 3 (low parasitemia), group B on day 5 (high parasitemia) and group C on day 15 (low parasitemia). Group D consisted of healthy rats (not-infected, n = 15) and were divided in three periods (n = 5) in order to compare with the infected groups. After euthanasia, cerebral cortex was removed for the preparation of synaptosomes and enzymatic assays. Group A showed no changes in enzymatic activities compared with control. The hydrolysis of ATP, ADP and AMP by the enzymes NTPDase and 5′-nucleotidase were increased (P < 0.05) in group B (38%, 140% and 61%, respectively) when compared with control. In the group C it was observed a decreased (22%) hydrolysis of ATP when compared with control group. The activities of NTPDase and 5′-nucleotidase in synaptosomes alters the acute phase of the disease when the number of circulating parasites is high, thus the change observed is probably due to the parasitemia.     

Phospholipid molecular species, beta-oxidation, desaturation and elongation of fatty acids in Atlantic salmon hepatocytes: effects of temperature and 3-thia fatty acids

We have investigated the effects of a 3-thia fatty acid (TTA) and of temperature on the fatty acid (FA) metabolism of Atlantic salmon (Salmo salar). One experiment investigated the activity of the peroxisomal beta-oxidation enzyme, acyl-CoA oxidase (ACO), and the incorporation of TTA into phospholipid (PL) molecular species. Salmon hepatocytes in culture were incubated either without TTA (control(spades)) or with 0.8 mM TTA (TTA(spades)) in a short term (48 h) temperature study at 5 degrees C and at 12 degrees C. TTA was incorporated into the four PL classes studied: phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI) and phosphatidylserine (PS). TTA was preferentially esterified with 18:1, 16:1, 20:4 and 22:6 in the PLs. Hepatocytes incubated with TTA had higher ACO activity at 5 degrees C than at 12 degrees C. In a second experiment salmon were fed a diet based on fish meal-fish oil without any TTA added (control) or a fish meal-fish oil diet supplemented with 0.6% TTA for 8 weeks at 12 degrees C and 20 weeks at 5 degrees C. At the end of the feeding trial, hepatocytes from fish acclimated to high or low temperatures were isolated from both dietary groups and incubated with either [1-(14)C]18:1 n-9 or [1-(14)C]20:4 n-3 at 5 degrees C or 12 degrees C. Radiolabelled 18:1 n-9 was mainly esterified into neutral lipids (NL), whereas [1-(14)C]20:4 n-3 was mainly esterified into PL at both temperatures. The rate of elongation of [1-(14)C]18:1 n-9 to 20:1 n-9 was twice as high in hepatocytes from fish fed the control diet than it was in hepatocytes from fish fed the TTA diet, at both temperatures. The amount of [1-(14)C]20:4 n-3 converted to 22:6 n-3 was approximately the same in hepatocytes from the two dietary groups, but there was a tendency to higher production of 22:6 n-3 at the lower temperature. Oxidation of [1-(14)C]18:1 n-9 to acid soluble products (ASP) and CO(2) was approximately 10-fold greater in hepatocytes kept at 5 degrees C than in those kept at 12 degrees C and the main oxidation products formed were acetate, oxaloacetate and malate.     

Cryptobia salmositica: susceptibility of infected rainbow trout, Salmo gairdneri, to environmental hypoxia

Using the sealed jar technique (also called residual oxygen bioassay), rainbow trout fry infected with Cryptobia salmositica were more susceptible than non-infected fish to environmental hypoxia. The Winkler technique (azide modification) was used to determine the residual dissolved oxygen in the water. Susceptibility of infected fish increased with 1) time after infection and was most evident in 3-7 wk infections, 2) the severity of anemia, and 3) increasing parasitemia. In prolonged infections, susceptibility was reduced when there were decreases in anemia and parasitemia; however, these infected fish were still more susceptible than non-infected fish. The increase in susceptibility of infected fish to hypoxia may be an important contributing factor to mortality of fish in hatcheries where there is inadequate water flow and overcrowding. The sealed jar technique is recommended in future studies on the pathogenesis of parasitic fish diseases, especially if the metabolic and/or respiratory systems are affected by the infection.     

New haemoglobin genotypes in Atlantic cod, Gadus morhua: possible relation with growth

In a preliminary study, 121 individually tagged juvenile Atlantic cod (Gadus morhua) were classified according to their haemoglobin genotypes into four groups, i.e., two main haemoglobin genotypes [Hb-I(1/2), Hb-I(2/2)] and two sub-types [Hb-I(1/2b), Hb-I(2/2b)], and reared for 3 months at 10 degrees C, 13 degrees C and T-step (fish reared at 16 degrees C and then subsequently moved to 13 and later to 10 degrees C). Overall growth rates across temperatures were 10% and 19% higher in the Hb-I(2/2b), Hb-I(1/2b) sub-types compared to corresponding Hb-I(2/2) and Hb-I(1/2) main types, respectively. Individual growth rate trajectories varied between the genotypes at all temperatures studied. Our study indicates that under certain environmental conditions higher growth in the two sub-types compared to the main genotypes could be expected. This may indicate difference in other physiological characters not studied here, but seen in previous studies, i.e., oxygen affinity and competitive performance.     

‘Therapeutic window’ for multiple drug treatment of experimental cerebral ischemia in gerbils

The effects of the following drugs: nimodipine (1 mg/kg b. w., i. p.), 2-amino-5-phosphonovaleric acid (4mg/kg b.w., i.p.) and propentofylline (25mg/kg b.w., i.p.), administered (alone or in combination) at the end of 15 min bilateral ischemia in gerbils were evaluated on mitochondrial superoxide dismutase (SOD), glutathione reductase (GR), glucose-6 phosphate dehydrogenase (G6PD), monoamine oxidase (MAO) activities, and thiobarbituric acid reactive material (TBARM), and brain water content at 1 hour of reperfusion. The combined treatment virtually abolished early postischemic brain edema (4.1% v.s. 0.6%) and efficiently counteracted ischemia-induced changes [decreased SOD (79% v.s. 98%), GR (52% v.s. 105%) and MAO (25% v.s. 79%), and increased TBARM (198% v.s. 108%)]. The same combination of drugs administered 15 min before ischemia had a similar effect (e.g., reduced brain swelling and lipid peroxidation) as when given at the end of ischemia, whereas a limited or absent impact was seen when the drugs were given 15 min or 1 hour after ischemia, respectively. The data suggest that (post)ischemic brain swelling and mitochondrial dysfunction can be reduced by drugs which synchronously prevent processes induced in the early stages of reperfusion.