Fish diseases endanger the aquaculture industry worldwide. Approaches to combat fish diseases are hampered by the relative poor knowledge of immune relevant genes in fish. This thesis describes the cloning and expression of several immune relevant genes in rainbow trout (Oncorhynchus mykiss). The full-length cDNA and genomic DNA of the inducible nitric oxide synthase (iNOS) gene have been cloned and sequenced. The gene structure was the first to be determined outwith the mammals. The expression of the iNOS gene is induced by virus (VHSV) infection, but its detection by RT-PCR might be impaired by the secondary structure formed in the iNOS mRNA. Screening of a genomic library resulted in the isolation of a second allele of IL-1b1 gene with a large intron III. This allele appears to have resulted from a recent retroposition of a HpaI SINE into intron III. Two other retroposition events have also been recognised in the same intron. The survival mechanism of this SINE is discussed and a model of trout IL-1b gene formation proposed. Both alleles of the IL-1b1 gene are constitutively expressed and induced by LPS and recombinant trout IL-1b1 in heterozygous RTS-11 cells. The promoter of the IL-1b1 allele S gene was isolated and the transcription start site was determined. A series of promoter-reportor gene constructs were transfected into RTG cells to study their activities under different conditions. The first intron is an important part of the promoter of IL-1b1 allele S and NFkB is a transcription factor required for IL-1b1 expression. The cytokine receptor common gamma chain (gC) was also isolated from rainbow trout, the first outwith mammals. Its expression can be detected in blood, gill, kidney, brain and liver, and in macrophage cultures. The expression of gC in macrophage cell cultures is upregulated by recombinant IL-1b1 and LPS stimulation. The expression of gC is also detectable in RTG-2 cells with an increased transcript level after stimulation with recombinant IL-1b1.

Flavobacterium psychrophilum is the causative agent of rainbow trout fry syndrome (RTFS) and bacterial cold water disease (BCWD) in salmonid fish world-wide. Basic information relating to the antigenic and biochemical characteristics, and pathogenicity of the bacterium are lacking in the literature. Therefore, the aim of this study was to characterise F. psychrophilum based on phenotypic and serological differences between isolates. The bacterium was also characterised by means of its extracellular products (ECPs). An attempt was made to develop an experimental challenge model for the bacterium. Phenotyping of the bacterium was based on growth and biochemical characteristics from which it was found that isolates of F. psychrophilum appeared homogenous. Intramuscular (IM) challenge was the most effective route for experimentally challenging rainbow trout fry with F. psychrophilum. Virulence of the bacterium was determined by injecting rainbow fry IM with different isolates of F. psychrophilum. Variations were found in the virulence of the different F. psychrophilum isolates when injected into fish by this route. The levels of protease activity and auto-agglutination characteristics appeared to vary between the virulent and non-virulent isolates. Electrophoretic analysis of whole cell preparations of F. psychrophilum showed that the protein and carbohydrate banding patterns of the different isolates were similar regardless of their origin or their virulence to rainbow trout. A substantial amount of carbohydrate was associated with the bacterium. Using a commercial glycoprotein detection kit, two glycoprotein bands were found at 20 and 23 kDa in whole cell preparations of the bacterium. The electrophoretic protein profiles of the outer membrane protein (OMP) preparations of the bacteria were similar between both virulent and non-virulent isolates. Characterisation of different F. psychrophilum isolates by an enzyme linked immunosorbent assay (ELlS A) using rabbit antisera raised against a virulent and nonvirulent isolate of F. psychrophilum, showed that there may be between three and five different serological groups. No association was detected between serotypes and geographical origin of the strains, the species of host fish from which they were recovered or the virulence of the isolates. The antisera detected common protein and carbohydrate antigens between the isolates with Western blot analysis. Antigenic differences were found between different F. psychrophilum isolates with ELISA and indirect fluorescent antibody technique (IF AT) using monoclonal antibodies (MAbs) developed against the virulent and the non-virulent F. psychrophilum isolates. Two MAbs (9H9 and 5A9) cross-reacted with a related species of bacterium F. branchiophilum, in the ELISA. Two MAbs (lE5 and 1 lB2) recognised high molecular weight material in whole cell preparations of the virulent F. psychrophilum in Western blot analysis, which also reacted with rainbow trout anti-F. psychrophilum sera raised against the virulent isolate of the bacterium. Due to their lack of specificity or sensitivity, both the rabbit sera and the eight MAbs produced in this study were considered unsuitable as diagnostic probes for screening infected RTFS samples. F. psychrophilum isolates produced varying amount of ECP proteins after 14 days culture in modified Anacker and Ordal's broth (MAOB), which exhibited substantial protease activity for casein and gelatin. However, the ECPs showed only partial haemolytic activity against rainbow trout eryhrocytes. Electrophoretic protein and Western blot profies were found to be very similar between the ECPs of different isolates. The ECP preparations contain glycoprotein molecules of either 20 or 23 kDa. None of the preparations from the virulent and the non-virulent isolates were found to be toxic to rainbow trout fry. The study suggests that isolates of F. psychrophilum are homogeneous in terms of their biochemical and electrophoretic characteristics, while antigenic characteristics varied between the isolates. The bacterium possesses a substantial amounts of carbohydrate and glycoprotein in its cellular and extracellular products.

A comprehensive survey of representative rainbow trout (Oncorhynchus mykiss) hatcheries revealed that the Gram negative, yellow-pigmented, filamentous bacterium Cytophaga psychrophila was implicated in a single disease in the U.K. and other European states. The involvement of C. psychrophila as the aetiological agent of the syndrome was substantiated by the fulfilment of Koch's postulates. Infectivity studies with isolates of C. psychrophila, carried out under natural and laboratory conditions successfully reproduced clinical signs and gross pathological changes analogous to those observed during field outbreaks of the condition. Histopathological examination of artificially and naturally infected fish tissues demonstrated several features that were considered to be pathognomonic for RTFS. Preliminary electron microscopic studies described the ultra-structure of C. psychrophila and partially elucidated the cellular response to the pathogen. Environmental sampling across selected hatchery sites demonstrated that members of the family Cytophagaceae formed a substantial element of the bacterial flora from natural waters, although recovery of C. psychrophila was restricted to areas where the substantial mortalities in fry attributable to RTFS had occurred. C. psychrophila was isolated from the sexual fluids of broodstock and additionally the bacterium was demonstrated associated with surfaces of eyed ova following various disinfection regimes. The minimum inhibitory concentrations of a range of antimicrobial agents both existing in, and novel to, aquaculture were examined, revealing compounds which would potentially mitigate losses attributable to RTFS during field outbreaks. The emergence of bacterial resistance to chemotherapeutants was discussed. The minimum inhibitory and bactericidal concentrations and required exposure times to a number of disinfecting agents were demonstrated. The efficacy of these agents as disinfectants of egg surfaces and equipment associated with fish production was assessed. The potential of a number of serodiagnostic techniques were evaluated as a means of rapid detection of C. psychrophila in diseased fish.

Temperature influences the incidence of chemically induced cancer in fish, with warmer temperatures being associated with higher cancer incidence. The mechanisms of temperature-modulated chemical carcinogenesis in fish, however, have not been described in detail. Therefore, one primary objective of this study was increased understanding of how temperature-modulated genotoxicity of 7,12- dimethylbenz(a)anthracene (DMBA) corresponded with tumor response. The second entails the potential of temperature to modulate cancer promotion. Rainbow trout (Oncorhyncus mykiss) (2 g) were acclimated at 10, 14 or 18��C for one month and then exposed to 1.0 ppm DMBA in their water for 20 hr. Exposures were at respective acclimation temperatures or 10 and 18��C acclimated fish were shifted to 14��C for DMBA exposures. Adduction of [��H]DMBA to hepatic DNA 21 days after exposure was higher in 10��C than 18��C fish exposed at their respective acclimation temperatures. However, in fish shifted to 14��C, the concentration of hepatic [��H]DMBA DNA adducts was similar in 10��C and 18��C acclimated fish at that time. Temperature effects on tumor incidence were assessed 9 months after DMBA waterborne exposures. Incidence of stomach, liver and swim-bladder cancer increased with rearing temperature. Differences in tumor incidence were less marked in fish reared at the same temperature (14��C). Retrospective analyses of livers from a tumor study initiated with aflatoxin B1 (AFB1) was conducted with antibodies to endogenous proliferating nuclear antigen (PCNA). Proliferating cells were scored by counting labeled nuclei in 5 random 10x fields using an image analysis program (BIOQUANT SYSTEM IV). There was no significant increase in numbers of PCNA labeled hepatocytes with temperature. The influence of acclimation temperature on plasma mitogens that stimulate cell division was assessed in cultured Chinook salmon embryo cells (CHSE-214). Plasma from rainbow trout (120-150 g) acclimated to either 10 or 18��C for at least four weeks stimulated in vitro proliferation of CHSE-214 cells to the same extent. This study demonstrated that chemically induced tumors in fish were modulated by temperature not only through genotoxin disposition and formation but also through persistence of DNA adducts. It also discounted the role of mitogenesis in temperature-modulated chemical carcinogenesis. / Graduation date: 1996

No description available.

Graduation date: 1994

Graduation date: 1996

The characteristics of an inhibitor of infectious pancreatic necrosis virus (IPNV) found in normal rainbow trout serum (RTS) were studied. The serum inhibitor had a molecular weight of approximately 150 kDa and was dependent on divalent cations, either Ca����� or Mg�����. It was stable at temperatures up to 50��C and at a pH range between 4-10. The inhibitor directly inactivated the virus and the inhibition level was dependent on cell densities and on the time at which virus was exposed to RTS. The level of virus inhibition by RTS was altered by the cell line in which virus was produced. IPNV was more efficiently inhibited by RTS in salmonid cell lines than in non-salmonid cell lines. Most of the salmonid sera tested showed inhibition, while non-salmonid sera did not inhibit IPNV replication. Rainbow trout continuously showed a significant level of inhibition in their serum after 23 weeks post hatch. Three isolates of IPNV were passaged five times in RTG-2 cells with either MEM-10 or MEM-10 with 1% rainbow trout serum and virus from each passage were tested for RTS sensitivity in vitro and virulence in vivo. The mortality level in brook trout fry was highly variable during viral passages, ranging between 30-89%. The RTS sensitivity and virulence were changed during viral passages, and these changes were dependent on cell culture conditions and IPNV isolate used. It was found that an IPNV crayfish isolate passaged in RTG-2 cells with MEM-10 showed significantly increased RTS sensitivity. This was, however, not correlated with decreased virulence. All three isolates showed identical antigenicity patterns with a panel of 11 monoclonal antibodies, irrespective of viral passage conditions. Clones prepared from an IPNV-Jasper (Ja) population which had been twice passed through brook trout were heterogeneous with respect to RTS sensitivity, serotype, and cDNA sequences. Eight percent of clones (4/50) were very sensitive to RTS (Ja-S), as was the parent strain, and eighty four percent of clones (42/50) showed a mid-range of RTS sensitivity. The final eight percent of clones (4/50) were RTS resistant (Ja-R). Enzyme immunodot assay revealed that Ja-S clones and Ja-R clones differed by several epitopes. Ja-S and Ja-R had significant differences in their cDNA sequences for the capsid protein VP2. These two strains shared 80.7% and 86% identity in nucleic acid and in amino acid sequences, respectively. / Graduation date: 2001

For ras gene mutation analysis in the rainbow trout (Oncorhynchus mykiss) model system, a partial trout ras sequence was identified using the polymerase chain reaction (PCR). Two synthetic oligonucleotides based on rat K-ras gene sequence were used as primers for the PCR procedure. A 90 base pair (bp) sequence, referred to as the trout K-ras, was amplified from trout genomic DNA and cDNA. Cloned 90 by PCR products from several normal liver tissues were sequenced resulting in the same sequence. Large-sized PCR products, 111 and 237 bp, were also cloned and sequenced indicating that these fragments included the 90 by sequence information expressed in mRNA. This 5'-terminal partial trout K-ras nucleotide sequence was 88% homologous to that of the goldfish ras gene, and less homologous to those of mammalian ras genes. Based on the partial sequence information of two trout ras genes, K-ras and H-ras, DNA from trout tumors induced by chemical carcinogens, aflatoxin B1 (AFB1) and N-methyl-N'-nitro-N-nitrosoguanidene (MNNG), were analyzed for the presence of point mutations. Using the PCR and oligonucleotide hybridization methods, a high proportion (10/14) of the AFB1-initiated liver tumor DNA indicated evidence for ras point mutations. Of the 10 mutant ras genotypes, seven were probed as G to T transversions at the second position of codon 12, two were G to T transversions at the second position of codon 13, and one was a G to A transition at the first position of codon 12. Nucleotide sequence analysis of cloned PCR products from four of these tumor DNAs provided definitive mutation evidence in each case, which seemed to occur in only a fraction of the neoplastic cells. However, no mutations were detected in exon 1 of the trout K-ras gene, nor in DNA from trout normal livers. Results indicated that the hepatocarcinogen AFB1 induced similar ras gene mutations in trout as in rat liver tumors. By comparison, the mutation specificity of MNNG in trout liver tumors was for G to A transitions, but no ras mutations were detected in trout kidney tumors. This investigation was the initial study of experimentally induced ras gene point mutations in a lower vertebrate fish model. / Graduation date: 1991

M.Sc. (Zoology) / Aphanomyces invadans is an oomycete associated with epizootic ulcerative syndrome (EUS). It affects more than a 100 freshwater and estuarine species of fish and is a serious threat to aquaculture and natural aquatic ecosystems. Currently, cases of EUS have been reported across Asia, Australia, North America and more recently Southern Africa. Outbreaks occur mostly during periods of sudden temperature change, such as heavy rainfall or change of seasons. These conditions favour sporulation of A. invadans, and low temperatures have been shown to delay the inflammatory response of fish to oomycete infection. Diagnosis of A. invadans is usually based on clinical signs and confirmed by demonstrating the presence of mycotic granulomas in histological section. Further diagnosis of EUS is made by isolation of A. invadans from internal tissues. Demonstrating typical asexual characteristics by inducing sporulation allows identification of the oomycete to the genus level. After inducing sporulation, the zoospores can be isolated for use in clinical infection of fish through subcutaneous injection or bath challenge systems. Standard molecular techniques such as polymerase chain reaction (PCR) have also been development for the fast and reliable diagnosis of the disease. The aim of this study was to perform different infection trials on EUS in two susceptible fish species, three-spot gourami, Trichogaster trichopterus, and rainbow trout, Oncorhynchus mykiss. Through initial trials, three-spot gourami was established as a suitable positive control species. Subsequently, hyphae were successfully re-isolated from infected fish and demonstrated as A. invadans, which was used in a further inoculation trial. Rainbow trout were challenged with A. invadans through intramuscular inoculation revealing varying degrees of susceptibility at different water temperatures. Detection of fungal hyphae and mycotic granulomas in tissue sections was achieved through histopathological examination, including the use of birefringence and fluorescents. Confirmation of A. invadans DNA in the various infection trials was done through PCR analyses. A histological grading system is proposed which will allow simplification of large scale qualitative microscopic analyses and identification of histological trends within a data set when analysing suspected cases of EUS. It is recommended that similar infection trials be applied to endemic species in Southern Africa to investigate their susceptibility to EUS.