Enhancement of phagocytic and chemokinetic activities of rainbow trout head kidney cells by C-reactive protein

OBJECTIVE: To investigate immunostimulating activity of purified rainbow trout (Oncorhynchus mykiss) C-reactive protein (CRP) on trout phagocytic cells. ANIMALS: 20 rainbow trout and 2 rabbits. PROCEDURE: The effect of CRP on phagocytic activity of head kidney (HK) cells was examined by use of a phagocytosis assay with plastic particles. The enhancing effect of CRP on migration activity of HK cells was examined by use of the blind well assay. RESULTS: Glass-adherent cells from clinically normal trout had increased dose-dependent phagocytic activity against plastic particles when cells were incubated in the presence of CRP. Pretreatment of particles with CRP also enhanced phagocytic activity of the cells, indicating an opsonic effect of CRP. Rabbit anti-trout CRP serum suppressed the enhancing activity of CRP. The HK cells had significant dose-dependent chemokinetic activity against CRP that was not inhibited by anti-CRP serum, indicating that a CRP-antibody complex also could be chemokinetic. CONCLUSIONS: Rainbow trout CRP has immunostimulating activity for HK cells, resulting in enhanced phagocytic and chemokinetic activities.     

Carbonic anhydrase in the utero-vaginal junction of immature and mature ostriches

1. Sperm storage tubules in the ostrich start to develop at an early stage of oviductal growth. Concurrently, membrane-bound carbonic anhydrase was found in the cells of the storage tubules. 2. In mature ostriches the utero-vaginal junction averaged 11.5+/-2.1 cm in length and primary mucosal folds were extremely long and slender. Membrane-bound carbonic anhydrase was present in the cells of the sperm storage tubules. In the non-ciliated cells of the surface epithelium both membrane-bound and cytoplasmic activity was detected. 3. The possible role of carbonic anhydrase in the stimulation/inhibition of sperm motility by altering the pH was discussed.     

一株虹鳟嗜冷益生菌Aeromonas popoffii的分离鉴定及体外抗病毒特性研究

为分离鉴定冷水鱼类的益生菌,本研究从健康的冷水鱼虹鳟幼鱼的肠道、胆囊等部位分离得到75株嗜冷耐低温生长菌株,经过耐低温温度梯度筛选及体外抗菌试验初步筛选出一株虹鳟嗜冷益生菌株,经革兰氏染色及API 50 CH生化鉴定、16S r DNA测序比对,鉴定为Aeromonas popoffii,将其命名为HL60分离株。通过耐胆酸游离酸试验、耐酸试验、体外抑菌试验和体外对鲤鱼上皮细胞(EPC)增殖影响试验及体外抗病毒(传染性造血器官坏死病病毒)试验,结果表明A.popoffii HL60分离株具有良好的益生特性及体外抗病毒活性。本研究分离获得的A.popoffii HL60分离株为冷水鱼益生菌株活载体疫苗开发及应用奠定了基础。     

Molecular regulation of lipid metabolism in liver and muscle of rainbow trout subjected to acute and chronic insulin treatments

Although the metabolic actions of insulin in fish have been investigated widely in the past several years, lipid metabolism has received little attention, especially in tissues like the liver or white muscle. In the present study, rainbow trout received insulin treatments both acutely (intraperitoneal injection) and chronically (through mino-osmotic pumps) to elucidate hormone metabolic actions at molecular levels on the 2 main insulin target tissues in trout, namely, liver and muscle. Plasma and free fatty acid concentrations in plasma, as well as mRNA measurements of some key enzymes involved in lipid metabolism, were assessed in these tissues after 6 h and 4 d of acute and chronic insulin treatments, respectively. Our results showed that although fish received the same final total amount of hormone in both treatments, the actions of insulin on lipid metabolism were both time and tissue dependent. After the acute insulin treatment, the main anabolic role of insulin was reflected in decreased plasma free fatty acid concentrations linked to enhanced hepatic lipogenesis. We also found that insulin increased the mRNA levels of enzymes involved in lipid oxidation, perhaps to counteract insulin-induced hypoglycemia. In contrast, our data show that after chronic insulin treatment, liver and muscle exhibit different metabolic strategies: whereas in the liver chronic insulin-induced hypoglycemia may stimulate lipolytic processes to spare glucose stores, the muscle responds directly to the anabolic hormone action by increasing its lipogenic capacity and by inhibiting pathways of lipid oxidation.     

Isolation and distribution of bacterial flora in farmed rainbow trout from Mexico

Trout farming is a growing aquaculture industry in Mexico, with stock mainly supplied by the importation of eyed eggs. The aim of the present study was to determine the frequency of bacterial isolations in farmed rainbow trout Oncorhynchus mykiss from Mexico. Sixty-five farms distributed among seven states of Mexico were included in the study. Individual samples from gills, liver, spleen, intestine, and kidney were obtained from 563 apparently healthy fish. In total, 371 bacterial isolates were recovered from sampled fish; isolates of the genera Aeromonas, Edwardsiella, Enterobacter, Escherichia, Klebsiella, Plesiomonas, Pseudomonas, and Yersinia were identified. Aeromonads were the most frequently isolated bacteria. Renibacterium salmoninarum was not isolated from any of the sampled fish. Our results showed the presence of bacteria that are potential pathogens of both rainbow trout and humans.     

Effect of alpha-tocopherol on antioxidant enzyme activities and lipid peroxidation in rainbow trout (Oncorhynchus mykiss)

In this study, the effect of alpha-tocopherol on the antioxidant capacity of rainbow trout (Oncorhynchus mykiss) was determined. For this purpose, activities of antioxidant enzymes such as catalase (CAT), peroxidase (POD) and glutathione reductase (GSSG-Rx) were investigated. Enzyme activities were measured at 0 (control), 1, 3 and 5 h after alpha-tocopherol injection. In addition, the levels of malondialdehyde (MDA) as a lipid peroxidation marker were determined in the erythrocytes. The results showed that alpha-tocopherol significantly activated the CAT, POD and GSSG-Rx enzymes as compared with the enzyme activities found in the controls (p < 0.05). However, MDA levels were significantly decreased by alpha-tocopherol treatment (p < 0.05). The results suggest that alpha-tocopherol may have a pro-oxidant tendency at a high dose and cause mild oxidative stress which could modulate signal transduction cascades, redirect gene expression, and influence many cellular responses such as proliferation, differentiation, and reproduction. For this reason, alpha-tocopherol should be used carefully in all applications in relation to fish.     

Immune complex-mediated glomerulonephritis associated with bacterial kidney disease in the rainbow trout (Oncorhynchus mykiss).

Rainbow trout (Oncorhynchus mykiss) developed a post-infectious chronic membranous glomerulonephritis 15 months after they had been experimentally infected with Renibacterium salmoninarum. Histologically, peritubular and periglomerular fibrosis, hypercellular glomeruli with occluded Bowman's space, and partial or complete adhesion to Bowman's capsule were constant features. Electron microscopy revealed thickened glomerular basement membranes with spikes accompanied by finely granular electron-dense deposits at the epithelial side and dense material in the mesangial matrix. Indirect immunofluorescence indicated linear immunoglobulin deposits along the glomerular basement membrane. The presence of R. salmoninarum was demonstrated by culture and by indirect immunofluorescence. Low serum hemagglutination-inhibiting antibody titers were demonstrated.     

Binding of Vibrio anguillarum to neutral glycosphingolipids from intestinal mucosa of rainbow trout (Oncorhynchus mykiss)

To test whether glycosphingolipids (GSLs) on the intestinal mucosa of rainbow trout (Oncorhynchus mykiss) serve as a binding receptor for Vibrio anguillarum, we analyzed neutral GSLs from rainbow trout intestinal mucosa and investigated the binding of bacteria to neutral GSLs. Two kinds of neutral GSLs, designated N-1 and N-2, were identified on high-performance thin-layer chromatography (TLC) plates. In TLC immunostaining tests, V. anguillarum bound only to galactosylceramide (GalCer), lactosylceramide and N-1 having the same TLC mobility as GalCer, but neither to glucosylceramide nor to N-2. These results suggest that N-1 is GalCer (Gal beta 1-1Cer) and also that N-1 (GalCer) on rainbow trout intestinal mucosa act as a receptor for V. anguillarum.     

Effect of vitamin E on carbonic anhydrase enzyme activity in rainbow trout (Oncorhynchus mykiss) erythrocytes in vitro and in vivo

Considering that the excessive usage of vitamin E causes hypervitaminosis and thus reduces blood erythrocyte concentrations, therefore it is worth studying how its pharmacological dosage affects the activity of carbonic anhydrase (CA) enzyme found in erythrocytes of rainbow trout (Oncorhynchus mykiss) in vitro and in vivo. Vitamin E inhibited CA enzyme and the IC50 value of the vitamin was 0.039 mM in vitro. Similarly, it was seen that vitamin E inhibited CA enzyme activity after the first hour following vitamin E injections in vivo. The activities of CA in groups of trout given vitamin E injection were measured at 1, 3 and 5 h and the corresponding activities were found to be 772.7 +/- 290.5 (P < 0.05), 1286.4 +/- 378.2 and 1005.7 +/- 436.1 enzyme units (EU) g Hb(-1). The difference over the control was significant (P < 0.05) in the first hour and insignificant at 3 and 5 h (P > 0.05). The activity of CA in the control, which did not contain vitamin E, was determined as 1597.7 +/- 429.0 EU g Hb(-1).     

Ultrastructural and clinicopathological studies on the toxicity of cationic acrylamide-based flocculant to rainbow trout

Rainbow trout (Salmo gairdneri) of two age groups were exposed to a cationic acrylamide-based flocculant at various concentrations in static bioassay chambers. At lethal concentrations the flocculant produced severe gill alterations in all fish. The principal alterations were necrosis and separation of the respiratory epithelial cells covering secondary lamellae. Many necrotic chloride cells were also seen, their apical plasma membrane was destroyed, and mitochondria were swollen with separated cristae. An influx of a large amount of fluid into the interstitial spaces caused partial or complete separation of subepithelial spaces from the covering epithelial cells and basement membranes of underlying blood vessels. Clinicopathological alterations included marked decreases in blood pH, partial pressure of oxygen, bicarbonate and plasma sodium, and chloride concentrations. Hematocrit, total protein, and blood glucose were increased. Fish exposed to sublethal concentrations had gill alterations characterized by hypercellularity and thickening of the secondary lamellae. These were due to undifferentiated cell proliferation and macrophage and lymphocyte infiltration between the covering epithelial cells and the underlying blood vessels. Macrophages and undifferentiated cells had large phagolysosomes containing cytoplasmic organelles, an indication of cell injury and increased turnover.     

Occurrence of enteric redmouth disease in rainbow trout (Oncorhynchus mykiss) on farms in Croatia

During the spring of 1996 and autumn of 1997 unusual mortality outbreaks among rainbow trout fry and yearlings occurred at two different trout farms, resulting in mortality of 20 and 10 per cent, respectively. Generally, the affected fish, swimming at the water surface, were reluctant to eat and were dark pigmented with visible haemorrhages around and within the oral cavity. Bacterial isolates from moribund fish from both cases were identified as Yersinia ruckeri by standard biochemical tests and API 20E. The isolated strains were found to be sensitive to tetracycline, chloramphenicol, co-trimoxazole, nalidixic acid, flumequine, enrofloxacin, carbenicillin and gentamicin. Microplate agglutination assay confirmed that both isolates belonged to serotype O1. The pathogenicity of the isolated bacteria was confirmed by challenge experiment. Titres of specific antibodies were determined in the sera of survivors. The titre was highest on the 21st day postchallenge and was detectable until the 81st day.     

The effects of Myxobolus cerebralis on the physiological performance of whirling disease resistant and susceptible strains of rainbow trout

The development of rainbow trout Oncorhynchus mykiss strains that are resistant to whirling disease has shown promise as a management tool for populations in areas where Myxobolus cerebralis is present. However, the physiological effects of the disease on characteristics necessary for fish survival in natural river conditions have not been tested in many of these strains. Five rainbow trout strains were evaluated for their swimming ability and growth characteristics in relation to M. cerebralis exposure: the resistant German rainbow trout (GR) strain (Hofer strain), the susceptible Colorado River rainbow trout (CRR) strain, and three intermediate (hybrid) strains (F1 = GR x CRR; F2 = F1 x F1; B2 = backcross of F1 x CRR). Three broad response patterns among strain and exposure were evident in our study. First, exposure metrics, growth performance, and swimming ability differed among strains. Second, exposure to the parasite did not necessarily produce differences in growth or swimming ability. Exposure to M. cerebralis did not affect batch weight for any strain, and critical swimming velocity did not differ between exposed and unexposed families. Third, although exposure did not necessarily affect growth or swimming ability, individuals that exhibited clinical deformities did show reduced growth and swimming performance; fish with clinical deformities were significantly smaller and had lower critical swimming velocities than exposed fish without clinical deformities. Research and management have focused on GR x CRR hybrid strains; however, given the performance of the GR strain in our study, it should not be discounted as a potential broodstock. Additional field trials comparing the GR and F1 strains should be conducted before wholesale adoption of the GR strain to reestablish rainbow trout populations in Colorado.     

Interaction of the attenuated recombinant rIHNV-Gvhsv GFP virus with macrophages from rainbow trout (Oncorhynchus mykiss)

One of the most important threats to the salmonid aquaculture industry is infection caused by novirhabdoviruses such as infectious haematopoietic necrosis virus (IHNV) or viral haemorrhagic septicaemia virus (VHSV). Using reverse genetics, an avirulent recombinant rIHNV-Gvhsv GFP strain was generated, which was able to replicate as effectively as wild type IHNV in a fish cell line and in macrophages. Although this recombinant virus induced protective responses against IHNV and VHSV, the response did not involve the production of antibodies or modulate the expression of some antiviral genes. To determine the immune mechanisms underlying the protection conferred by the rIHNV-Gvhsv GFP virus, different immune parameters (NO production, respiratory burst activity and the induction of apoptosis) were assessed in the macrophage population. The results obtained in the present work may indicate that the Nv protein could be important in the modulation of NO and ROS production. rIHNV-Gvhsv GFP did not appear to have a clear effect on nitric oxide production or apoptosis. However, an increased respiratory burst activity (with levels induced by the recombinant virus significantly higher than the levels induced by the wild type virus), suggests a stimulation of the macrophage population, which could be related to the protection against virulent viruses.     

Purification and partial characterization of a cadmium-binding protein from the liver of rainbow trout (Onchorynchus mykiss).

This study describes the isolation and partial characterization of a low molecular weight (approximately 14 kDa), cadmium-binding protein from rainbow trout (Onchorynchus mykiss) liver. Rainbow trout were injected intraperitoneally with 3.5 mg/kg cadmium chloride (total body dose) twice weekly for 3 wk. Livers were removed and a cadmium-binding protein was isolated. Monoclonal antibodies produced against this protein were used in the affinity purification process. Amino acid analysis showed the protein contained 3.8 mol% cysteine, 3.5 mol% phenylalanine, 2.2 mol% tyrosine and 1.9 mol% histidine. The low cysteine content suggests that it was distinct from metallothionein. The monoclonal antibodies were also used to identify the protein in liver homogenates from both cadmium-exposed and control fish and in the testes of cadmium-exposed mice lacking the gene for both metallothionein-1 and metallothionein-II. The compound identified in this study represents a non-metallothionein cadmium-binding protein that appears to be highly conserved.     

Detection of early stages of Myxobolus cerebralis in fin clips from rainbow trout (Orynchus mykiss).

A nested polymerase chain reaction (PCR) assay was used to detect early stages of Myxobolus cerebralis in caudal and adipose fin samples from rainbow trout (RT). To determine sensitivity, groups of 10 RT were exposed to 2,000 M. cerebralis triactinomyxons/fish for 1 hour at 15 degrees C and subsequently moved to clean recirculating water. Fish were held for 2 and 6 hours and 1, 2, 3, 5, 7, 10, 30, and 60 days before sampling by nonlethal fin biopsy. Nested PCR performed on fin clips showed that M. cerebralis DNA was detected in caudal fin tissue in 100% of fish up to 5 days postexposure. At days 7 and 10 postexposure, 80% of fish were positive, and at 60 days postexposure, 60% of fish were positive using this technique. Conversely, testing on adipose fin clips proved less sensitive, as positive fish dropped from 80% at day 7 to below 20% at day 10 postinfection. Since detection of M. cerebralis infection using caudal fin samples coupled with nested PCR is an effective method for detection of early parasite stages, use of this technique provides for accurate, nonlethal testing.     

Effect of florfenicol on the immune response of rainbow trout (Oncorhynchus mykiss)

Florfenicol, a drug effective against several bacterial diseases of fish, was tested for possible immunomodulatory effects. The aim of the study was to follow the kinetics of the immune response after vaccination with simultaneous oral antibiotic treatment. The fish were immunised with a commercial oil-based divalent (furunculosis/vibriosis) vaccine and were simultaneously given oral antibiotic treatment. The specific immune response was monitored by analysing the levels of specific antibodies with ELISA. As an indicator of the non-specific immune response the phagocytic activity of circulating leucocytes was measured by a chemiluminescence assay. Total circulating leucocyte counts and differentials were also monitored. The disease resistance was evaluated by challenge tests at the end of the experiment. The results showed that florfenicol did not have any significant effect on antibody production and circulating leucocyte levels but caused a suppression in chemiluminescence response/phagocytic cell 5-6 weeks after vaccination. The survival after challenge was slightly suppressed by the florfenicol treatment. The RPS-value for the vaccinated group was 98% and for the florfenicol-treated group was 88%.     

在豆粕型饲料中添加蛋氨酸羟基类似物(MHA)对虹鳟鱼生产性能的影响

试验2本试验的目的是研究MHA在豆粕型实用配方中的最适添加量。