Y.M. Sin

Development and maturation of the immune system in zebrafish, Danio rerio: a gene expression profiling, in situ hybridization and immunological study.

The development and maturation of the immune system in zebrafish was investigated using immune-related gene expression profiling by quantitative real-time polymerase chain reaction, in situ hybridization (ISH), immunoglobulin (Ig) detection by immuno-affinity purification and Western blotting as well as immersion immunization experiments. Ikaros expression was first detected at 1 day post-fertilization (dpf) and thereafter increased gradually to more than two-fold between 28 and 42dpf before decreasing to less than the initial 1dpf expression level in adult fish (aged 105dpf). Recombination activating gene-1 (Rag-1) expression levels increased rapidly (by 10-fold) between 3 and 17dpf, reaching a maximum between 21 and 28dpf before decreasing gradually. However, in adult fish aged 105dpf, the expression level of Rag-1 had dropped markedly, and was equivalent to the expression level at 3dpf. T-cell receptor alpha constant region and immunoglobulin light chain constant region (IgLC) isotype-1, 2 and 3 mRNAs were detected at low levels by 3dpf and their expression levels increased steadily to the adult range between 4 and 6 weeks post-fertilization (wpf). Using tissue-section ISH, Rag-1 expression was detected in head kidney by 2wpf while IgLC-1, 2 and 3 were detected in the head kidney and the thymus by 3wpf onwards. Secreted Ig was only detectable using immuno-affinity purification and Western blotting by 4wpf. Humoral response to T-independent antigen (formalin-killed Aeromonas hydrophila) and T-dependent antigen (human gamma globulin) was observed in zebrafish immunized at 4 and 6wpf, respectively, indicating that immunocompetence was achieved. The findings reveal that the zebrafish immune system is morphologically and functionally mature by 4-6wpf.

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.

Development of a tropical marine fish cell line from Asian seabass (Lates calcarifer) for virus isolation

A tropical marine fish cell line (SF) was established from Asian seabass (Lates calcarifer) fry. The cell line was maintained in Earles minimum essential medium (EMEM) supplemented with foetal calf serum and incubated at 25°C. The susceptibility of the cell-line at the 16th, 35th and 46th subculture to several iridoviruses, birnaviruses, reoviruses, a rhabdovirus, and a nodavirus was studied. Cytopathic effect (CPE) was observed daily after virus inoculation and viral replication efficiency was determined for six viruses. SF cell cultures infected with an iridovirus, a nodavirus and a reovirus were further elucidated by electron microscopy. All the viruses tested were shown to induce CPE on SF cells. This suggests that the SF cell line has good potential for the isolation of various fish viruses. The SF cell line consists predominantly of epithelial-like cells and has been subcultured 85 times over a period of 24 months.

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.

Some virulence characteristics of Aeromonas hydrophila in walking catfish (Clarias gariepinus)

Abstract Aeromonas hydrophila isolated from diseased walking catfish from West Java, Indonesia was studied for its virulence using the following parameters: biochemical characteristics, presence of haemolysin genes by DNA-DNA hybridization, haemolytic activity, protein analysis by SDS-PAGE and LD50. The results showed that many strains of Aeromonas hydrophila with differing virulence are present in the aquatic environment of this region. The virulent strains possess haemolysin genes and showed a 52-kDa band in SDS gel. The bacteria could lyse erythrocytes not only from fish but also from sheep, man, cow, horse and rabbit. They caused severe skin and muscle lesion at the injected sites of catfish fingerlings and remained in their ulcerative tissues, liver and kidney until 5 days after intramuscular injection. Fish began to die at 18 h after the bacterial injection. It is concluded that the outbreak of such disease in walking catfish in Java in 1980 could be attributed to these virulent strains of Aeromonas hydrophila.

Passive transfer of protective immunity against ichthyophthiriasis from vaccinated mother to fry in tilapias, Oreochromis aureus

Abstract Tilapias, Oreochromis aureus , were vaccinated twice with live tomites of Ichthyophthirius multifiliis in physiological saline 1 month before spawning (test group). A control group was similarly treated but with physiological saline only. Fertilized eggs were collected from the mouths of both groups and incubated until the young reached the free-swimming stage. Free-swimming fry were also collected immediately after they were expelled from the mothers mouth in other fish of the two groups. The fry were exposed to infective tomites of I. multifiliis and thereafter returned to their holding tanks. All fry obtained from the controls without mouth-brooding died after tomite exposure. However, fry with mouth-brooding before tomite exposure showed 37.3% survival. On the other hand, fry obtained from vaccinated broodstock without mouth-brooding exhibited 78.4% survival which increased to 95.3% with mouth-brooding. The protective immunity is correlated with the titres of anti- I. multifiliis antibodies in the soluble extracts of fry tissues and the mothers plasma. The results clearly indicate that protective immunity against ichthyophthiriasis “ich” in tilapia fry can not only be derived directly from the mother via eggs but also be acquired indirectly from the mouth cavity during the brooding period. The results thus suggest that proper vaccination of mothers before spawning would effectively protect fry against a protozoan disease.

Orthogonal array design as a chemometric method for the optimization of analytical procedures. Part 4. Mixed-level design and its application to the high-performance liquid chromatographic determination of polycyclic aromatic hydrocarbons

The theory and methodology of a mixed-level orthogonal array design for the optimization of analytical procedures is described. Firstly, the method of construction of a mixed-level orthogonal array design is discussed in detail, followed by the assignment of experiments and the data analysis strategy in the design. The optimization of the parameters involved in the high-performance liquid chromatographic determination of polycyclic aromatic hydrocarbons is then employed to demonstrate the practical application of the proposed mixed-level orthogonal array design in the area of analytical chemistry.

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.