Diplostomiasis, or eyefluke is caused by the metacercarial stage of the digenean parasite <i>Diplostomum spathaceum</i> which selectively invades the lenses of freshwater fish and can cause partial and total blindness. This study investigates the host-parasite relationship of <i>D. spathaceum</i> infections in rainbow trout with particular reference to the diplostomule or post-penetration stage of the parasite. The point of cercarial penetration influenced the speed with which diplostomules reached the lens and the length of time they were exposed to the fish defence mechanisms. The further away the point of cercarial penetration from the eye, the longer diplostomules take to reach the lens and the fewer establish as metacercariae. Total body immersion using cercariae resulted in significantly more metacercariae establishing in the lens compared with entry via intramuscular, intraperitoneal or intracardial injection of either cercariae or diplostomules. Cercariae may be drawn in by the respiratory current and enter the fish via the gills and buccal cavity. The gills provide an immediate source of entry into the circulatory and lymphatic system of the fish. There did not appear to be any preferred route of migration. The optimum conditions for the <i>in vitro</i> collection of diplostomules were developed. The optimum conditions for <i>in vitro</i> transformation of <i>D. spathaceum</i> cercariae were established. The optimum conditions for the short-term <i>in vitro</i> maintenance of diplostomules were investigated. L-15 medium supplemented with 5% heat-inactivated foetal calf serum maintained approximately 80% survival for the first 24-48 h of culture. Rainbow trout were immunized with cercariae and diplostomules of <i>D. spathaceum</i>. Circulating antibody was detected by ELISA to both cercariae and diplostomules by 6-7 weeks post-immunization. Cross-reactivity occurred between anti-cercarial serum and diplostomule and metacercarial antigens, and anti-diplostomule serum cross-reacted with cercarial and metacercarial antigens in ELISA. Immunofluoresence studies indicated that the tail region of the cercriae was strongly antigenic; a differential fluorescence was observed for both anti-cercarial and anti-diplostomule sera with the cercarial tails fluorescing more strongly than the bodies. No cross-reactivity was observed in fluorescence between anti-cercarial or anti-diplostomule sera and metacercarial antigen although anti-cercariae serum cross-reacted with diplostomule antigen and <i>vice versa</i>. Normal trout serum and anti-diplostomule serum were cytotoxic to diplostomules of <i>D. spathaceum in vitro</i>. Immunized fish exhibited a significant degree of protection against further infection by <i>D. spathaceum</i>. However, there was no correlation between protection and the level of serum antibody. Isolated normal trout macrophages kill significant numbers of diplostomules <i>in vitro</i> at effector: parasite ratios of 150:1 or greater. <i>In vitro</i> killing was not increased using diplostomules opsonized with anti-serum or <i>in vivo</i>-activated macrophages individually, but when combined, increased killing occurred. Diplostomules were capable of eliciting respiratory burst activity from macrophages <i>in vitro</i>, suggesting that reactive oxygen species may have a role to play in the killing mechanism. Diplostomule antigen was capable of activating macrophages <i>in vivo</i>.

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To develop robust and reproducible methods for molecular typing of <I>Toxoplasma </I>strains, the DNA regions of 5<I>S</I> rDNA, 28S-18S rDNA <I>IGS SAG2</I>, and <I>GRA6</I> loci were examined. The 5<I>S</I> sequences were identical among 24 different strains; sequencing of the <I>IGS</I> region showed a few polymorphisms (0.66%) distinguishing virulence types. The IGS PCR-RFLP methods were developed and used to examine 29 strains of different virulence types. Sequence analysis of the IGS 5'-end showed great diversity between <I>Neospora caninum </I>and <I>T. gondii. </I>The IGS-RFLPs also clearly distinguished between those two closely related species. Nucleotide sequencing of the <I>SAG2</I> locus (a surface antigen coding gene) showed 1.37% polymorphisms among 24 strains. Apart from a single nucleotide change at the 5'-flanking region, the type III and type I strains were identical. However, three new alleles of this locus were identified in minor variants of the strains. Analysis of the coding region of the <I>GRA6</I> locus (a dense granule antigen coding gene) revealed a great degree of polymorphisms (3.24%) among 33 strains. Nine different alleles, representing the three current types and the minor variants of strains were characterised at this locus. A PCR-RFLP based on <I>GRA6</I> polymorphisms was developed which could distinguish the three major types of <I>T. gondii</I>. This marker proved to be a suitable tool for a population study of the <I>Toxoplasma </I>parasite. The predominance of non-synonymous nucleotide substitutions in <I>SAG2</I> and <I>GRA6</I> genes confirmed positive selection in these loci, suggesting they play an important role in the parasite virulence. Phylogenetic analysis based on the multi-locus sequence alignment showed the existence of more than three lineages in <I>Toxoplasma </I>populations.

A standardised procedure was established for the laboratory maintenance of C.irritans in thick-lipped mullet. Nine isolates of C.irritans were obtained of which eight were successfully established for up to 48 weeks. Studies on individual life cycle stages with regards to longevity, viability, and susceptibility to a chemotherapeutic agent, revealed the difficulties in eradicating the cysts. Transmission of the parasite both to and from the host correlated with darkness. High infection levels resulted in the death of host fish within five days following exposure to theronts. An acquired protective immune response developed in host mullet within 14 days after exposure to sub-lethal infection. The degree of immunity appeared to relate to infection dose, and was not fully protective in all fish. Protection persisted for six months after infection and appeared specific to C.irritans. Specific antibodies to trophont antigen were identified in mullet serum but not epithelial mucus following either natural exposure to theronts or intraperitoneal immunisation with trophont antigens. Serum from intraperitoneally immunised fish caused theront immobilisation and agglutination in vitro; however no evidence was found for a protective role for specific antibody. Major polypeptides were identified and characterised by molecular weight for both trophont and theront stages using SDS-PAGE. Significant homology in major polypeptide profiles was found between C.irritans and I.multifiliis, in respect to trophonts and particularly theronts. Murine monoclonal antibodies raised to trophonts identified two polypeptide components of molecular weights 20-21kDa and 68-69kDa, the latter being homologous with host immunoglobulin heavy chain. These results are discussed in relation to future management and control strategies for cryptocaryosis in warmwater mariculture systems and aquaria.

The ability of thick-lipped grey mullet to mount both humoral and cellular immune responses to Cryptocotyle lingua has been demonstrated here for the first time. Of the 3 procedures for immunisation investigated, namely exposure to live cercariae, intraperitoneal (ip) injection of whole killed cercariae and ip injection of a cercarial sonicate, the former gave the highest primary antibody titres with a peak response at 4 weeks post infection. Peak titres occurred at 5 and 7 weeks after immunisation by ip injection of sonicated cercariae and whole killed cercariae respectively. An enhanced secondary antibody response was observed following challenge by ip injection of sonicated cercariae. Cercariacidal activity, apparently involving complement, was detected in normal serum and enhanced in immune serum. However, the mechanism for complement activation remains uncertain. Parasite antigens were identified following separation by SOS-PAGE and immunoblotting using mullet and rabbit antisera. Mullet failed to respond to certain parasite proteins recognised by rabbit antiserum and in situ immunogold-silver staining demonstrated that mullet. immunised by exposure to live cercariae, were unable to recognise the metacercarial cyst wall. Migration and polarisation of pronephric leucocytes in vitro was enhanced following immunisation but cellular adherence to encysted and unencysted parasites was not observed. In vitro studies further demonstrated a possible role for fish serum in stimulating metacercarial encystment. The intensity of melanisation of the host capsule in metacercarial infections was increased in fish held in total darkness and with increased temperature. The rate of development of the metacercarial tegument and cyst wall and the associated host response was similarly affected by temperature and initial development was inhibited by prior immunisation via ip injection of sonicated cercariae. There was however, no evidence of protection against cercarial infection following such immunisation. These results are discussed in relation to mechanisms of immunity, metacercarial survival strategies and control of metazoan infections in fish.

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