Prolonged in vitro cultivation of Ichthyophthirius multifiliis using an EPC cell line as substrate.
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The ciliate Ichthyophthirius multifiliis, which normally requires a fish host to develop from the theront stage to the trophont stage, was cultivated in vitro for part of its life cycle. Experiments were conducted using a laboratory strain of the parasite originally isolated from rainbow trout Oncorhynchus mykiss in a Danish trout farm. Theronts escaping from tomontocysts were kept in water, cell culture media (E-MEM or L-15), or cultures of EPC (Epithelioma Papulosum Cyprini) cells in plastic tissue culture dishes (Nunc multidish plates). In addition, a 2-compartment system, with water separated from tissue culture media by a monolayer of EPC cells on an Anopore Tissue Culture Insert (mimicking the fish epidermis) was tested as an experimental habitat for the parasite. Theronts transformed into trophonts in all treatments except in water alone. However, development was accelerated in wells containing EPC cells, and survival and growth of trophonts were significantly increased compared to water or tissue culture media alone. Further, the 2-compartment system allowed superior performance of the parasites (attachment of parasites to cells and growth from 36 to 46 microm). In all experiments it was found that the presence of host factors (mucus and serum) stimulated parasite development. (+info)
Uptake and processing of biofilm and free-cell vaccines of Aeromonas hydrophila in indian major carps and common carp following oral vaccination--antigen localization by a monoclonal antibody.
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Uptake and processing of biofilm (BF) and free-cell (FC) vaccines of Aeromonas hydrophila were studied in the Indian major carps catla Catla catla, and rohu Labeo rohita and in the common carp Cyprinus carpio following a single dose oral vaccination of 10(11) CFU g(-1) fish. Fish were sampled at 0.5, 1, 3, 6, 12, 24 h and at 2, 3, 5 and 10 d following vaccination and antigen localization was studied in the gut, kidney and spleen employing monoclonal antibody based immunofluorescence and immunoperoxidase. The general pattern of antigen localization was similar in catla, rohu and common carp. Initially, both the BF and FC antigens were localized in the gut lumen, followed by their uptake by intraepithelial vacuoles and macrophages. Antigen administered orally was also seen in the spleen and kidney. Both BF and FC antigens were detected in the gut lumen of carp within 30 min following oral delivery. However, BF antigen remained in the lumen of the hindgut for 48 h compared to 6 h in the case of FC antigen. In the early stages, BF antigen was localized in the gut epithelial vacuoles while FC antigen was associated with the small macrophages of the hindgut. Antigen localization in spleen and kidney was observed at 3 h and persisted even up to 10 d following oral delivery. In general, there was a distinct difference between BF and FC vaccines in the duration of retention and quantity of uptake in the gut, kidney and spleen. (+info)
Does anoxia induce cell swelling in carp brains? In vivo MRI measurements in crucian carp and common carp.
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Although both common and crucian carp survived 2 h of anoxia at 18 degrees C, the response of their brains to anoxia was quite different and indicative of the fact that the crucian carp is anoxia tolerant while the common carp is not. Using in vivo T(2) and diffusion-weighted magnetic resonance imaging (MRI), we studied anoxia induced changes in brain volume, free water content (T(2)), and water homeostasis (water diffusion coefficient). The anoxic crucian carp showed no signs of brain swelling or changes in brain water homeostasis even after 24 h except for the optic lobes, where cellular edema was indicated. The entire common carp brain suffered from cellular edema, net water gain, and a volume increase (by 6.5%) that proceeded during 100 min normoxic recovery (by 10%). The common carp recovered from this insult, proving that the changes were reversible and suggesting that the oversized brain cavity allows brain swelling during energy deficiency without a resultant increase in intracranial pressure and global ischemia. It is tempting to suggest that this is a function of the large brain cavity seen in many ectothermic vertebrates. (+info)
Edwardsiella tarda mutants defective in siderophore production, motility, serum resistance and catalase activity.
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Edwardsiella tarda is a Gram-negative bacterium that causes a systemic infection, edwardsiellosis, in fish. The virulence factors of this pathogen and its genetic determinants have not been systematically examined. In this study, TnphoA transposon mutagenesis was used to construct a library of 440 alkaline phosphatase (PhoA(+)) fusion mutants from a total of 400000 transconjugants derived from Ed. tarda PPD130/91. This library included genes for secreted and membrane-associated proteins normally involved in virulence. The library was screened for four virulence factors: siderophore production, motility, serum resistance and catalase production. Eight mutants deficient in one or more of these phenotypes were grouped into four classes. They were further characterized for their stimulation of reactive oxygen intermediate production by fish phagocytes, for their adhesion to and internalization into EPC (epithelioma papillosum of carp) cells, and for attenuation of virulence in blue gourami. Mutants 2A and 34 were highly attenuated in fish, with LD(50) values about 10 times higher than for the wild-type. These strains had mutations in the genes encoding arylsulfate sulfotransferase (mutant 2A) and a catalase precursor protein (mutant 34). One hyperinvasive/adhesive mutant and four pst mutants that were pleiotropic and slightly attenuated in fish were also isolated. (+info)
Slow PIII component of the carp electroretinogram.
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The slow PIII component of the electroretinogram (ERG) was studied in the isolated, aspartate-treated carp retina. Although the latter is richly populated with cones, slow PIII appeared to reflect almost exclusively the activity of rods; e.g. the spectral sensitivity of the potential paralleled closely the rod pigment curve, its operating range (i.e. the V-log I curve) was limited to 3 log units above absolute threshold, and raising background intensities to photopic levels produced saturation of the increment threshold function without evidence of a cone-mediated segment. Only after bleaching away a significant fraction of the porphyropsin was it possible to unmask a small photopic contribution to slow PIII, as evidenced by a displacement in the action spectrum to longer wavelengths. The spatial distribution of the slow PIII voltage within the retina (Faber, D.S. 1969. Ph.D. Thesis. State University of New York. Buffalo, N.Y.; Witkovsky, P.J. Nelson, and H. Ripps. 1973. J. Gen Physiol. 61:401) and its ability to survive aspartate treatment indicate that this potential arises in the Muller (glial) fiber. Additional support for this conclusion is provided by the slow rise time (several seconds) and long temporal integration (up to 40s) of the response. In many respects the properties of slow PIII resemble those of the c-wave, a pigment epithelial response also subserved by rod activity. On the other hand, the receptoral (fast PIII) and the b-wave components of the ERG behave quite differently. Unlike slow PIII, response saturation could not be induced, since both potentials are subserved by cones when the stimulus conditions exceed the limits of the scotopic range. Receptors appear to govern light adaptation at photopic background levels; both fast PIII and b-wave manifest identical incremental threshold values over this range of intensities. However, under scotopic conditions, the sensitivity of the b-wave is affected by luminous backgrounds too weak to alter fast PIII threshold, indicating a postreceptoral stage of adaptation. (+info)
Long-term cortisol treatment inhibits pubertal development in male common carp, Cyprinus carpio L.
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The onset and regulation of puberty is determined by functional development of the brain-pituitary-gonad (BPG) axis. Stress has been shown to interfere with reproduction and the functioning of the BPG axis. The response to chronic and severe stress may require much energy and force the organism to make adaptive choices. Energy that is normally available for processes like growth, immune response, or reproduction will be channeled into restoration of the disturbed homeostasis. Cortisol plays a key role in the homeostatic adaptation during or after stress. In the present study, immature common carp were fed with cortisol-containing food pellets covering the pubertal period. We showed that cortisol caused an inhibition of pubertal development, by affecting directly or indirectly all components of the BPG axis. The salmon GnRH content of the brain was decreased. Luteinizing hormone- and FSH-encoding mRNA levels in the pituitary and LH plasma levels were diminished by long-term cortisol treatment, as was the testicular androgen secretion. Testicular development, reflected by gonadosomatic index and the first wave of spermatogenesis, was retarded. (+info)
Unique biochemical nature of carp retinol-binding protein. N-linked glycosylation and uncleavable NH2-terminal signal peptide.
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Retinol transport and metabolism have been well characterized in mammals; however, very little is known in fish. To study the mechanism by which fish retinol-binding protein (RBP) is able to remain in plasma besides its small molecular size, we isolated RBP cDNA from a carp liver cDNA library. Comparison of the deduced amino acid sequence with that of known vertebrate RBPs showed that carp RBP has high homology to the other cloned vertebrate RBPs, but it lacks the COOH-terminal tetrapeptide, RNL(S)L, which is most likely involved in the interaction with transthyretin in mammalian RBPs. In addition, the primary structure of carp RBP contains two consensus N-linked glycosylation sites that represent a unique feature. We have obtained experimental evidence, by in vitro and in vivo expression experiments, that both sites are indeed glycosylated. We have also characterized the protein as a complex type N-linked glycoprotein by lectin binding assay, neuraminidase and endoglycosidase H and F digestion. Inhibition of glycosylation by tunicamycin treatment of transfected cells caused a great reduction of RBP secretion. Since kidney filtration of anionic proteins is less than half that of neutral protein of the same size, this finding strongly suggests that the amount of carp RBP filtration through kidney glomeruli may be reduced by a glycosylation-dependent increase in the molecular size and negative charge of the protein. A second unique feature of carp RBP as secretory protein is the presence of a nonconserved NH(2)-terminal hydrophobic domain, which functions as an insertion signal but is not cleaved cotranslationally and remains in the secreted RBP. (+info)
Identification of beta-endorphins in the pituitary gland and blood plasma of the common carp (Cyprinus carpio).
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Carp beta-endorphin is posttranslationally modified by N-terminal acetylation and C-terminal cleavage. These processes determine the biological activity of the beta-endorphins. Forms of beta-endorphin were identified in the pars intermedia and the pars distalis of the pituitary gland of the common carp (Cyprinus carpio), as well as the forms released in vitro and into the blood. After separation and quantitation by high performance liquid chromatography (HPLC) coupled with radioimmunoassay, the beta-endorphin immunoreactive products were identified by electrospray ionisation mass spectrometry and peptide sequencing. The release of beta-endorphins by the pituitary gland was studied after stimulation with corticotrophin-releasing factor (CRF) in vitro. In the pars intermedia, eight N-acetylated truncated forms were identified. Full length N-acetyl beta-endorphin(1-33) coeluted with N-acetyl beta-endorphin(1-29) and these forms together amounted to over 50% of total immunoreactivity. These products were partially processed to N-acetyl betaendorphin(1-15) (30.8% of total immunoreactivity) and N-acetyl beta-endorphin(1-10) (3.1%) via two different cleavage pathways. The acetylated carp homologues of mammalian alpha- and gamma-endorphin were also found. N-acetyl beta-endorphin(1-15) and (1-29) and/or (1-33) were the major products to be released in vitro, and were the only acetylated beta-endorphins found in blood plasma, although never together. CRF stimulated the release of opioid beta-endorphin from the pars distalis. This non-acetylated beta-endorphin represents the full length peptide and is the most abundant form in plasma. (+info)