Toong Jin Lam

The effects of crowding stress on the non-specific immuneresponse in fancy carp (Cyprinus carpio L.)

Abstract The effect of crowding condition (25g 1 −1 ) on the non-specific immune response of fancy carp ( Cyprinus carpio L.) was studied. The fish were stressed for 1, 7, 14 and 30 days. The total serum protein concentration, serum lysozyme activity, bactericidal complement activity and chemiluminescent (CL) response of the phagocytic pronephros cells were all measured on day 30 after the initial crowding stress. Their susceptibility to infectious disease was tested by challenging with the bacterium Aeromonas hydrophila . Stress indicators such as plasma cortisol, glucose and chloride levels were also determined. The results indicated that the levels of cortisol and glucose were significantly increased from day 1 onwards until day 30. On the other hand, the activities of non-specific immune parameters except for phagocytic activity significantly dropped on day 1, and then remained low throughout the remaining time intervals. Disease resistance of the stressed fish was significantly reduced on day 7. However, no great difference in resistance against A. hydrophila was found between day 7 and day 30. No mortality was encountered in either stressed or unstressed fish. This indicates that fish might have an adaptation capability to survive under a stable, chronically stressful environment although the activities of non-specific defence mechanisms were significantly lower than those of the unstressed fish.

Electron microscopic observations of a marine fish iridovirus isolated from brown-spotted grouper, Epinephelus tauvina.

The morphogenesis and the ultrastructure of a marine fish iridovirus isolated from diseased grouper, Epinephelus tauvina were studied by electron microscopy. The virus was grown on a marine fish cell line (GP) at 25 degrees C. After appearance of advanced cytopathic effect (CPE), various morphogenetic stages of virus amplification, maturation and assembly were detected in the cytoplasm of virus-infected cells. The matured nucleocapsids were probably formed by insertion of electron-dense core material into a partly forming empty capsid just before completely sealed. The nucleocapsids were located at the assembly sites as pseudocrystalline arrays or scattered individually. In the late phase of infection, the nucleocapsids were enveloped and released by budding from the plasma membrane. The budding virus particles could directly enter neighbouring cells by endocytosis to start the next round infection. Ultrastructure of the grouper iridovirus was studied using the methods of enzymatic digestions and detergent degradations. The purified iridovirus particles showed a three-layered membrane including an external lipoprotein envelope, an inner periodic protein capsid and a lipid-containing membrane. The regular array of surface capsid subunits was observed after degradation with detergent.

Faithful expression of green fluorescent protein (GFP) in transgenic zebrafish embryos under control of zebrafish gene promoters

Although the zebrafish has become a popular model organism for vertebrate developmental and genetic analyses, its use in transgenic studies still suffers from the scarcity of homologous gene promoters. In the present study, three different zebrafish cDNA clones were isolated and sequenced completely, and their expression patterns were characterized by whole-mount in situ hybridization as well as by Northern blot hybridization. The first clone encodes a type II cytokeratin (CK), which is specifically expressed in skin epithelia in early embryos and prominently expressed in the adult skin tissue. The second clone is muscle specific and encodes a muscle creatine kinase (MCK). The third clone, expressed ubiquitously in all tissues, is derived from an acidic ribosomal phosphoprotein P0 (arp) gene. In order to test the fidelity of zebrafish embryos in transgenic expression, the promoters of the three genes were isolated using a rapid linker-mediated PCR approach and subsequently ligated to a modified green fluorescent protein (gfp) reporter gene. When the three hybrid GFP constructs were introduced into zebrafish embryos by microinjection, the three promoters were activated faithfully in developing zebrafish embryos. The 2.2-kb ck promoter was sufficient to direct GFP expression in skin epithelia, although a weak expression in muscle was also observed in a few embryos. This pattern of transgenic expression is consistent with the expression pattern of the endogenous cytokeratin gene. The 1.5-kb mck promoter/gfp was expressed exclusively in skeletal muscles and not elsewhere. By contrast, the 0.8-kb ubiquitous promoter plus the first intron of the arp gene were capable of expressing GFP in a variety of tissues, including the skin, muscle, lens, neurons, notochord, and circulating blood cells. Our experiments, therefore, further demonstrated that zebrafish embryos can faithfully express exogenously introduced genes under the control of zebrafish promoters.

CLONING AND CHARACTERIZATION OF VASCULAR ENDOTHELIAL GROWTH FACTOR (VEGF) FROM ZEBRAFISH, DANIO RERIO

We have cloned and sequenced a zebrafish (Danio rerio) Vascular Endothelial Growth Factor (vegf) cDNA. It encodes a precursor protein of 188 amino acids with a putative 23 amino acids signal peptide. Sequence comparison analysis indicates that the zebrafish vegf cDNA corresponds to the human VEGF165 isoform and shows about 52% identity to human VEGF165 at the amino acid level. A 2.8 kb vegf message RNA was detected in adult zebrafish by Northern blot analysis. Expression of vegf165 is also detected by RT-PCR in adult fish and throughout the zebrafish embryonic development. Whole mount in situ hybridization of zebrafish embryos indicates strong expression in four areas of the 18-19 h post-fertilization (hpf) embryo: within the anterior central nervous system in the prospective optic stalk, in mesoderm overlapping the bilaterally located merging heart fields, in mesoderm underlying and flanking the hindbrain posterior to rhombomere 4, and in medial regions of the somites. The study of vegf function in zebrafish embryonic vascular development will contribute to our understanding of the mechanisms of vertebrate endothelial cell differentiation and vasculature formation.

Nodavirus infection in freshwater ornamental fish, guppy, Poicelia reticulata--comparative characterization and pathogenicity studies.

Summary. Biochemical, genomic and serological studies were carried out to characterize a virus obtained from diseased guppy, Poicelia reticulata. The SDS-PAGE analysis of CsCl purified virus showed two distinct bands with molecular weight of 42 kDa and 110 kDa. A 1367 nucleotide region of the coat protein gene was sequenced, which includes one full open reading frame of 1017 nucleotides and a region of 350 nucleotides at the 3′end. The nucleotide identity of this strain with the nodavirus isolated from Epinephelus tauvina (Singapore strain) is 98% and with other strains of fish nodaviruses the identity is more than 75%. Western blot analysis using rabbit antisera raised against the nodavirus from marine fish, E. tauvina confirmed its antigenic similarity to the marine nodavirus isolate. Asymptomatic infection in guppy fry was observed following experimental infection with this virus and the marine nodavirus isolate (Singapore strain) implying the spread of virus from marine fish to freshwater fish. This report forms the first description of a nodavirus infection in freshwater fish.

Characterization, pathogenicity and neutralization studies of a nervous necrosis virus isolated from grouper, Epinephelus tauvina, in Singapore

Abstract A virus isolated from diseased marine fish, Grouper, Epinephelus tauvina, was cultured in sea bass (SB) cell line, characterized and its pathogenicity and neutralization studies were carried out. This isolated virus is an icosahedral virus with a mean diameter of 28–30 nm and has buoyant density of 1.30–1.35 g/ml. It replicates exclusively in the cytoplasm and forms paracrystalline array and inclusion bodies in the infected cells. SDS-PAGE analysis of purified virus structural proteins resolved one major polypeptide of approximately 42 kDa. This virus induces cytopathic effects such as rounding and granulation of cells, localized cell death and detachment of cells within 3–5 days postinfection on sea bass larval cell line. Typical histopathological changes in the virus-infected sea bass larvae under experimental conditions showed vacuolation of nervous tissue. Polyclonal antisera raised against the purified virus and the coat protein of this virus effectively neutralized virus infectivity in vitro suggesting the use of coat protein as a vaccine against this viral infection.

Antigenic characterization of a marine fish iridovirus from grouper, Epinephelus spp

Iridoviruses, recognized as causative agents of serious systemic diseases, have been identified from more than 20 fish species. Antigenic properties of a pathogenic iridovirus isolated from grouper, Epinephelus spp., in Singapore (SGIV) were investigated using rabbit IgG against the virus. Antisera were prepared by immunization of rabbit with purified virions. The rabbit IgG was purified from antiserum using a protein A-agarose column and adsorbed onto acetone-dried grouper (GP) cells. The viral surface-exposed antigens were visualized by a combination of immunogold transmission electron-microscopy and by indirect immunofluorescence, and the viral antigenic related proteins were discriminated by Western blot. The cross immunofluorescence assay showed that the grouper virus isolate was serologically close to viruses of the genus Ranavirus of family Iridoviridae. The viral antigens were detected from virus infected-cell cultures as early as 4 h of post infection using IFAT, and could be detectable in virus-infected fish blood as early as 3 days post infection. Immuno-dot assays revealed that the rabbit anti-SGIV IgG allowed sensitive detection of SGIV viral antigens. This study will facilitate the development of diagnostic techniques and vaccines for grouper iridovirus.

Protection of goldfish against Ichthyophthirius multifiliis by immunization with a recombinant vaccine

Abstract A 316 bp gene fragment containing a potential antigenic epitope of the 48 kDa immobilization antigen of Ichthyophthirius multifiliis (i-AgI) was assembled from six synthetic oligonucleotides and cloned into a bacterial expression vector pGEX2T. The gene construct was introduced into Escherichia coli and the glutathione S-transferase-iAgI (GST-iAgI) fusion protein was successfully expressed. Antisera against GST-iAgI fusion protein from catfish showed a positive reaction with a tomites protein of about 48 kDa, suggesting that the recombinant protein contains an antigenic epitope of i-AgI. Naive goldfish that were immunized with purified GST-iAgI fusion protein were challenged with a lethal dose of infectious tomites of I. multifiliis. The results showed that the average survival rate of the immunized fish was 95% as compared to 55% for the control fish. All these findings suggest that the recombinant GST-iAgI fusion protein can be used as a potential vaccine against the infection of I. multifiliis.

Temperature dependence of estrogen binding: importance of a subzone in the ligand binding domain of a novel piscine estrogen receptor.

The full length estrogen receptor from Oreochromis aureus (OaER) was cloned and expressed in vitro and in vivo as a functional transcription factor. Amino acid residues involved in the thermal stability of the receptor are located at/near subzones beta1 and beta3, which are highly conserved in other non-piscine species but not in OaER. Hormone binding studies, however, indicate that OaER is thermally stable but exhibited a approximately 3-fold reduced affinity for estrogen at elevated temperatures. Transfection of OaER into various cell lines cultured at different temperatures displayed a significant estrogen dose-response shift compared with that of chicken ER (cER). At 37 degrees C, OaER requires approximately 80-fold more estrogen to achieve half-maximal stimulation of CAT. Lowering of the incubation temperature from 37 degrees C to 25 degrees C or 20 degrees C resulted in a 4-fold increase in its affinity for estrogen. The thermally deficient transactivation of OaER at temperatures above 25 degrees C was fully prevented by high levels of estrogen. Thus, compared to cER, the OaER exhibits reduced affinity for estrogen at elevated temperature as reflected in its deficient transactivation capability. Amino acid replacements of OaER beta3 subzones with corresponding amino acids from cER could partially rescue this temperature sensitivity. The three-dimensional structure of the OaER ligand binding domain (LBD) was modelled based on conformational similarity and sequence homology with human RXRalpha apo, RARgamma holo and ERalpha LBDs. Unliganded and 17beta-estradiol-liganded OaER LBD retained the overall folding pattern of the nuclear receptor LBDs. The residues at/near the subzone beta3 of the LBD constitute the central core of OaER structure. Thus, amino acid alteration at this region potentially alters the structure and consequently its temperature-dependent ligand binding properties.

Generation of Two-color Transgenic Zebrafish Using the Green and Red Fluorescent Protein Reporter Genes gfp and rfp

Abstract: Two tissue-specific promoters were used to express both green fluorescent protein (GFP) and red fluorescent protein (RFP) in transgenic zebrafish embryos. One promoter (CK), derived from a cytokeratin gene, is active specifically in skin epithelia in embryos, and the other promoter (MLC) from a muscle-specific gene encodes a myosin light chain 2 polypeptide. When the 2 promoters drove the 2 reporter genes to express in the same embryos, both genes were faithfully expressed in the respective tissues, skin or muscle. When the 2 fluorescent proteins were expressed in the same skin or muscle cells under the same promoter, GFP fluorescence appeared earlier than RFP fluorescence in both skin and muscle tissues, probably owing to a higher detection sensitivity of GFP. However, RFP appeared to be more stable as its fluorescence steadily increased during development. Finally, F1 transgenic offspring were obtained expressing GFP in skin cells under the CK promoter and RFP in muscle cells under the MLC promoter. Our study demonstrates the feasibility of monitoring expression of multiple genes in different tissues in the same transgenic organism.