Lipopolysaccharide O-antigen prevents phagocytosis of Vibrio anguillarum by rainbow trout (Oncorhynchus mykiss) skin epithelial cells

Colonization of host tissues is a first step taken by many pathogens during the initial stages of infection. Despite the impact of bacterial disease on wild and farmed fish, only a few direct studies have characterized bacterial factors required for colonization of fish tissues. In this study, using live-cell and confocal microscopy, rainbow trout skin epithelial cells, the main structural component of the skin epidermis, were demonstrated to phagocytize bacteria. Mutant analyses showed that the fish pathogen Vibrio anguillarum required the lipopolysaccharide O-antigen to evade phagocytosis and that O-antigen transport required the putative wzm-wzt-wbhA operon, which encodes two ABC polysaccharide transporter proteins and a methyltransferase. Pretreatment of the epithelial cells with mannose prevented phagocytosis of V. anguillarum suggesting that a mannose receptor is involved in the uptake process. In addition, the O-antigen transport mutants could not colonize the skin but they did colonize the intestines of rainbow trout. The O-antigen polysaccharides were also shown to aid resistance to the antimicrobial factors, lysozyme and polymyxin B. In summary, rainbow trout skin epithelial cells play a role in the fish innate immunity by clearing bacteria from the skin epidermis. In defense, V. anguillarum utilizes O-antigen polysaccharides to evade phagocytosis by the epithelial cells allowing it to colonize rapidly fish skin tissues.     

Bacterial skin flora variation and in vitro inhibitory activity against Saprolegnia parasitica in brown and rainbow trout

Variations in the number and diversity of bacteria from the skin of brown trout Salmo trutta L. and rainbow trout Oncorhynchus mykiss Walbaum were surveyed from different rivers and fish farms in northern Spain. In addition to determining bacterial populations in skin samples of healthy fish, bacterial populations were determined from skin lesions (of brown trout only) infected with Saprolegnia parasitica, the causal agent of saprolegniosis. Mean bacterial counts from skin lesions of brown trout suffering from saprolegniosis were nearly 1000 times greater than from the skin of uninfected brown and rainbow trout. More than 20 different genera of bacteria were identified, with isolates of Aeromonas and Iodobacter being the predominant genera associated with saprolegniosis lesions. The in vitro inhibitory activity of 72 of these skin isolates was tested against S. parasitica using 3 different assays. These included (1) assessing the inhibition by bacteria of colony growth on agar media, (2) the inhibition of colony growth from colonized hemp seeds in liquid media and (3) the inhibition of cyst germination in liquid media. Finally, the fungicidal effect of the 24 most inhibitory bacterial species, and the inhibitory activity of their culture supernatants, was tested in the same way. Isolates identified as Aeromonas piscicola, A. sobria, Pantoea agglomerans and Pseudomonas fluorescens achieved the highest inhibition against S. parasitica. Many of these inhibitory isolates were obtained primarily from skin lesions of fish with saprolegniosis. It is suggested that some of these isolates might be useful in the biological control of saprolegniosis.     

Identification of a molecular marker for type A spermatogonia by microarray analysis using gonadal cells from pvasa-GFP transgenic rainbow trout (Oncorhynchus mykiss)

The spermatogonia of fish can be classified as being either undifferentiated type A spermatogonia or differentiated type B spermatogonia. Although type A spermatogonia, which contain spermatogonial stem cells, have been demonstrated to be a suitable material for germ cell transplantation, no molecular markers for distinguishing between type A and type B spermatogonia in fish have been developed to date. We therefore sought to develop a molecular marker for type A spermatogonia in rainbow trout. Using GFP-dependent flow cytometry (FCM), enriched fractions of type A and type B spermatogonia, testicular somatic cells, and primordial germ cells were prepared from rainbow trout possessing the green fluorescent protein (GFP) gene driven by trout vasa regulatory regions (pvasa-GFP rainbow trout). The gene-expression profiles of each cell fraction were then compared with a microarray containing cDNAs representing 16,006 genes from several salmonid species. Genes exhibiting high expression for type A spermatogonia relative to above-mentioned other types of gonadal cells were identified and subjected to RT-PCR and quatitative PCR analysis. Since only the rainbow trout notch1 homologue showed significantly high expression in the type A spermatogonia-enriched fraction, we propose that notch1 may be a useful molecular marker for type A spermatogonia. The combination of GFP-dependent FCM and microarray analysis of pvasa-GFP rainbow trout can therefore be applied to the identification of potentially useful molecular markers of germ cells in fish.     

Expression patterns of gdnf and gfrα1 in rainbow trout testis

In mice, glial cell line-derived neurotrophic factor (GDNF) is essential for normal spermatogenesis and in vitro culture of spermatogonial stem cells. In murine testes, GDNF acts as paracrine factor; Sertoli cells secrete it to a subset of spermatogonial cells expressing its receptor, GDNF family receptor α1 (GFRα1). However, in fish, it is unclear what types of cells express gdnf and gfrα1. In this study, we isolated the rainbow trout orthologues of these genes and analyzed their expression patterns during spermatogenesis. In rainbow trout testes, gdnf and gfrα1 were expressed in almost all type A spermatogonia (ASG). Noticeably, unlike in mice, the expression of gdnf was not observed in Sertoli cells in rainbow trout. During spermatogenesis, the expression levels of these genes changed synchronously; gdnf and gfrα1 showed high expression in ASG and decreased dramatically in subsequent developmental stages. These results suggested that GDNF most likely acts as an autocrine factor in rainbow trout testes.     

Changes in apoptotic rate and cell viability in three fish epidermis cultures after exposure to nonylphenol and to a wastewater sample containing low concentrations of nonylphenol

Three types of epidermal cultures of fish were used for toxicological investigations, a primary cell culture and a tissue culture prepared from the rainbow trout Oncorhynchus mykiss Walbaum and the cell line EPC, derived from a skin tumour of the carp Cyprinus carpio L. Two studies were carried out to compare the different culture systems. In the first cultures were incubated with nonylphenol and in the second set of experiments the cell cultures were exposed to a wastewater sample containing low concentrations of nonylphenol (NP). Both cell cultures were similarly sensitive to nonylphenol with respect to the endpoints cell viability (LC₅₀     

Long-term primary culture of epithelial cells from rainbow trout (Oncorhynchus mykiss) liver

Summary Long-term primary cultures of epithelial cells from rainbow trout (Oncorhynchus mykiss) liver have been established. Nearly homogenous (>97%) populations of hepatocytes were placed into primary culture and remained viable and proliferative for at least 70 d. In addition to hepatocytes, proliferative biliary cells persisted in the cultures for at least 30 d. Finally, a third type of epithelial cell, which we have termed a “spindle cell,” consistently appeared and proliferated to confluence in these cultures. The confluent cultures of spindle cells were successfully subcultured and passaged. The initial behavior, growth, and optimization of serum and media requirements for these cells is described. All three cell types proliferated as measured by thymidine incorporation, autoradiography, proliferating cellular nuclear antigen analysis, and propidium iodine staining. Further efforts to characterize the cells included western blotting and immunohistochemical staining with antibodies to cytokeratins previously reported in fish liver. From these data, it appears that all three cell populations are epithelial in nature. Furthermore, significant changes in actin organization, often indicative of transformation or pluripotent cells, were observed with increased time in primary culture.     

The environmental impact of salmonid cage culture on inland fisheries: present status and future trends

This paper briefly reviews the environmental impact of the cage culture of salmonids in fresh waters, and evaluates the effects of this fish farming practice on the growth, behaviour and diseases of inland fish populations, concentrating on relationships between rainbow trout cage culture and indigenous brown trout populations. Possible future developments in salmonid cage culture are also discussed and the implications of this development for inland fisheries evaluated. The paper argues that the development of models should enable improved prediction of eutrophication from cage culture, to the benefit of the fish farmer, inland fisheries and other freshwater users, but also identifies a clear need for research in other areas of possible conflict.     

Influence of storage temperature on infectious hematopoietic necrosis virus detection by cell culture isolation and RT-PCR methods

The detection of infectious hematopoietic necrosis virus (IHNV) in infected rainbow trout Oncorhynchus mykiss and in cell culture supernatants stored under different conditions was studied. IHNV-positive fish visceral organ homogenates and cell culture supernatants were incubated at 4 and 25 degrees C. Virus titre was measured by virus isolation on epithelioma papulosum cyprini (EPC) cells and the IHNV RNA was detected by RT-PCR and semi-nested RT-PCR. The influence of repeated freezing and thawing on the virus isolation from organ homogenates and from cell culture supernatants was studied as well. It was possible to isolate the virus from IHNV-positive organ material during the 3 d of incubation at 4 degrees C but, only on the first day of incubation at 25 degrees C. Viral RNA could be amplified during the incubation period of 35 d at 4 degrees C but only during 8 d of incubation at 25 degrees C. In IHNV-infected cell culture supernatant stored at 4 degrees C, it was possible to detect virus for 36 and 16 d in supernatant stored at 25 degrees C. Viral RNA could be followed by using molecular methods during the entire experimental period of 123 d. Each cycle of freezing and thawing of samples resulted in a reduction of IHNV titre in the suspension of visceral organs, while the virus titre in cell culture supernatant remained almost the same following 33 freezing-thawing cycles. The present results show that rapid laboratory processing and storage of potentially virus-containing tissue samples as well as the use of different detection methods are very important for efficient IHNV diagnosis.     

Occurrence of Flavobacterium psychrophilum in fish-farming environments

Occurrence of Flavobacterium psychrophilum in fish farms and fish-farming environments was studied using agar plate cultivation, the immunoflourescence antibody technique (IFAT) and nested PCR. Characteristics of 64 F. psychrophilum isolates from rainbow trout Oncorhynchus mykiss, fish farm rearing water, ovarian fluid and wild fish were serotyped, ribotyped and compared biochemically. Virulence of F. psychrophilum isolates from different sources was compared by injection into rainbow trout. Additionally, the number of F. psychrophilum cells shed by naturally infected rainbow trout was determined. F. psychrophilum was detected and isolated from skin mucus, skin lesions and internal organs of diseased rainbow trout and from fish without clinical disease. The pathogen was also present in wild perch Perca fluviatilis, roach Rutilus rutilus, and ovarian fluids of farmed rainbow trout brood fish. Isolates were biochemically homogenous, excluding the capability to degrade elastin. Five different agglutination patterns with different antisera against F. psychrophilum were found among the isolates studied. Although several different ribopatterns were found (ClaI: 12 ribopatterns and HaeIII: 9 ribopatterns), ribotype A was the most dominant. Farmed rainbow trout brood fish carried a broad-spectrum of serologically and genetically different F. psychrophilum in ovarian fluids. Virulence of the tested isolates in rainbow trout varied and naturally infected rainbow trout shed 10(4) to 10(8) cells fish(-1) h(-1) of F. psychrophilum into the surrounding water.     

Characterization of morphology and physiological actions of scale osteoclasts in the rainbow trout

The morphology of scale osteoclasts in rainbow trout Oncorhynchus mykiss was characterized by light and scanning electron microscopy, and the effects of oestradiol-17β-treatment and sexual maturation on scale osteoclast morphology were investigated. The cells associated with resorption cavities could be distinguished morphologically as two types: symmetrical, compact cells lacking or having only a few cell processes, termed type 1 cells, and asymmetrical cells covered with folds and having several cell processes, termed type 2 cells. In adult sexually maturing fish, where scale resorption was high, type 1 cells were predominant. In juveniles and spawned adults where scale resorption was assumed to be relatively low, mostly type 2 cells were present. Oestradiol 17-β-treatment of juvenile rainbow trout increased the osteoclast activity, but did not affect the osteoclast morphology. Using light microscopy, the majority of the cells observed in, and closely associated with, the resorption cavities were mononucleated in both maturing and spawned fish. Occasionally, bi- and multinucleated osteoclasts were observed in the maturing, but not in the spawned fish. Light microscopic enzyme-histochemistry showed that the majority of the mononucleated cells, as well as the bi- and multinucleated ones, were tartrate resistant acid phosphatase positive in both groups of fish, thus implying that both type 1 and type 2 cells were osteoclasts. It is thus apparent that scale resorption in rainbow trout is carried out by two morphologically distinct osteoclast populations, representing different stages of osteoclast activity and/or different stages of osteoclast differentiation.     

Viability and differential function of rainbow trout liver cells in primary culture: Coculture with two permanent fish cells

Summary The study investigates the influence of different culture conditions on attachment, viability and functional status of rainbow trout (Oncorhynchus mykiss) liver cells in primary culture. Cells were isolated by a two-step collagenase perfusion and incubated in serum-free, chemically defined minimal essential medium (MEM), (a) as a monolayer on uncoated PRI-MARIA? dishes, (b) as a monolayer on culture dishes coated with calf collagen type 1, and (c) in coculture with the established fish cell lines RTH-149 or RTG-2. Cell attachment was assessed from DNA and protein concentrations per dish, viability was estimated from cellular lactate dehydrogenase release, and the metabolic status was investigated by measuring activities of the phosphoenolpyruvate carboxykinase and biotransformation enzymes as well as the total cytochrome P450 contents. Seeding of hepatocytes on collagen-coated dishes did not alter cell attachment or detachment from the culture substrate, but had a small, but not significant effect on cell viability and metabolic parameters. Coculture of liver cells and RTG-2 cells reduced hepatocyte detachment from the culture substrate, and it was associated with a significant elevation of 7-ethoxyresorufin-O-deethylase activities in the hepatic cells. Cytochrome P450 contents, however, were not altered. The coculture effect on liver cell physiology clearly depended on the type of cell line, because coculture with RTH-149 cells led to similar, but much weaker effects than obtained in cocultures with RTG-2 cells. Electron microscopical observations revealed the existence of gap junctions and possible exocytosis-like transport between cell lines and hepatocytes. The results point to the potential of coculture systems to improve physiological parameters of trout liver cells in primary culture.     

Characterization of primary culture of rainbow trout (Oncorhynchus mykiss) skin explants: Growth, cell composition, proliferation, and apoptosis

A trout (Oncorhynchus mykiss) epidermal skin primary explant system was evaluated over 8 d by light and electron microscopy. Three distinct regions of the explant outgrowth were identified on the basis of cell composition. The area immediately adjacent to the founder tissue contained mainly small migrating cells and mucous cells. Of the former. about 20% were mitotic and 6% apoptotic. The middle area was characterized by differentiated pavement cells and mucous cells, with fewer small migrating cells. Proliferation was approximately 30% and apoptosis 5%. Over time, total cell numbers halved as more pavement cells differentiated. The growing front contained many mucous and small migrating cells initially, with few pavement cells. About 50% of the cells were in the proliferative phase, and 5% were apoptotic. Later, there were fewer migrating and mucous cells, with a higher number of pavement cells. About 9% of the cells were apoptotic, and 70% of the cells were proliferating. As in vivo, pavement cells had apical microridges, although they were vacuolated and contained phagocytosed apoptotic bodies. The data and observations are based on the numbers of cell cultures prepared from separate trout giving the sample size n = 7. As this culture system is reproducible and closely approximates the epidermis of trout, it is a powerful tool to study the effects of pollutants, parasites, and endocrine factors on fish skin, eliminating whole-animal factors and reducing the number of experimental animals required.     

DNA repair synthesis in cultured fish and human cells exposed to fish S9-activated aromatic hydrocarbons

Unscheduled DNA repair synthesis was measured autoradiographically in cultured rainbow trout gonad (RTG) and human fibroblast (HF) cells following exposure to aflatoxin B1 (AFB1), 3,4-benzopyrene (BP), 1,2,5,6-dibenzanthracene (DBA), 1,2-benzanthracene (BA) and pyrene (PY) activated with S9 prepared from rainbow trout liver. S9 from rainbow trout injected with Arochlor 1254 or an oil extract was compared with S9 from Fischer rats injected with Arochlor 1254 for the ability to activate AFB1 and cause DNA repair in RTG and HF cells. All three types of S9 activated AFB1, but the measured DNA repair response was greater in the HF cells. A significant grain count response was found following exposure of HF cells to fish S9-activated BP. Using assay conditions which enhance fish cell grain counts, a significant level of DNA repair was also found in RTG cells exposed to fish S9-activated BP. Marginal but statistically significant amounts of DNA repair were elicited in HF and RTG cells exposed to rainbow trout S9-activated BA and DBA, but no response was detected following PY exposure. Fish S9 was found to be able to activate a series of polycyclic aromatic hydrocarbons (PAH) and cause DNA repair synthesis in both fish and mammalian cells. The magnitude of the repair response roughly parallels the carcinogenic potential of the PAHs. These results elicit trans species and phyla comparisons which help to validate fish as models for aquatic carcinogenesis research, and also demonstrate PAH DNA-damaging effects on fish DNA, adding further credence for studying the effects of these chemicals on aquatic organisms.     

A stromal cell line from rainbow trout spleen, RTS34st, that supports the growth of rainbow trout macrophages and produces conditioned medium with mitogenic effects of leukocytes

Summary A rainbow trout spleen cell line, RTS34, was developed from a long-term hemopoietic culture. This cell line consisted of a mixed stromal cell layer with an associated cell population of macrophage-like cells that formed proliferative foci and released nonadherent progeny cells into the culture medium. A stromal cell line, RTS34st, was isolated from the RTS34 cell line. RTS34st cultures contained cells with fibroblast-like and epithelial-like morphologies and showed enhanced [³H]thymidine incorporation in response to either FBS or rainbow trout serum. The combination of FBS and trout serum was synergistic. Conditioned medium from RTS34st stimulated thymidine incorporation by peripheral blood and head kidney leukocytes, but not by leukocytes from the spleen. In addition, RTS34st provided a hemopoietic inductive microenvironment for immature precursor cells, selectively supporting the growth of macrophage-like cells. Therefore, RTS34st appears useful for studying the different roles of the stroma in regulating hemopoiesis in fish.     

Genetic (co)variation in skin pigmentation patterns and growth in rainbow trout

From a physiological-behavioral perspective, it has been shown that fish with a higher density of black eumelanin spots are more dominant, less sensitive to stress, have higher feed intake, better feed efficiency and therefore are larger in size. Thus, we hypothesized that genetic (co)variation between skin pigmentation patterns and growth exists and it is advantageous in rainbow trout. The objective of this study was to determine the genetic relationships between skin pigmentation patterns and BW in a breeding population of rainbow trout. We performed a genetic analysis of pigmentation traits including dorsal color (DC), lateral band (LB) intensity, amount of spotting above (SA) and below (SB) the lateral line, and BW at harvest (HW). Variance components were estimated using a multi-trait linear animal model fitted by restricted maximum likelihood. Estimated heritabilities were 0.08±0.02, 0.17±0.03, 0.44±0.04, 0.17±0.04 and 0.23±0.04 for DC, LB, SA, SB and HW, respectively. Genetic correlations between HW and skin color traits were 0.42±0.13, 0.32±0.14 and 0.25±0.11 for LB, SA and SB, respectively. These results indicate positive, but low to moderate genetic relationships between the amount of spotting and BW in rainbow trout. Thus, higher levels of spotting are genetically associated with better growth performance in this population.     

虹鳟补体活化调节因子CD46的分子特征及功能

为进一步研究硬骨鱼类中补体活化调节因子的分子特征和功能,研究克隆了虹鳟(Oncorhynchus mykiss)的CD46基因,对其分子特征进行了系统分析,结果显示:虹鳟CD46基因由10个外显子和9个内含子组成,cDNA序列全长2812 bp,编码317个氨基酸,蛋白序列由1个信号肽、4个SCR结构域、1个跨膜区和1个胞内区组成,预测分子量为33.9 kD。基因组共线性分析显示,虹鳟CD46基因位于16号染色体,其基因座在脊椎动物中具有保守的共线性。组织和白细胞亚群表达分析显示,虹鳟CD46基因在各种组织和白细胞亚群中均有表达。为了进一步阐明虹鳟CD46的免疫功能,研究原核表达纯化了标签蛋白GST和融合蛋白GST-CD46。溶血活性实验表明,与GST相比, GST-CD46能够显著抑制虹鳟血清对兔红细胞的溶血活性,且呈现剂量依赖效应,表明虹鳟CD46是补体活化的调节因子。此外,研究用HEK293T细胞过表达了GFP和GFP-CD46。细胞损伤实验显示,与GFP相比, GFP-CD46能够显著抑制虹鳟血清对HEK293T细胞的损伤,进一步表明虹鳟CD46是补体活化的调节因子,能够保护细...     

Skin mucus protease from rainbow trout, Salmo gairdneri Richardson, and its biological significance

The skin mucus of rainbow trout, salmon, charr, cod, coalfish, plaice and redfish contain, besides lysozyme and haemagglutinins, protease activity. The mucus protease from rainbow trout has been purified, characterized and shown to be indistinguishable from the trypsin produced in pylorus caecae. This enzyme activity can escape detection in mucus since it is inhibited by serum components which may contaminate the skin mucus as a result of stress and handling of the fish prior to collecting the mucus.