Studies on host responses to Aphanomyces invadans
Aphanomyces invadans is the pathogen that causes epizootic ulcerative syndrome (EUS), an economically devastating fish disease in southern Asia. The present thesis considered possible improvements to current methods of monitoring EUS, and examined the mechanisms of the host immune response to A. invadans in order to establish whether they could be enhanced to reduce the impact of EUS on aquaculture. Monoclonal antibody (MAb) technology was considered as a possible improvement to the histopathological methods currently used in diagnosis of EUS. Five MAbs were raised to day-old A. invadans germlings. Four gave weak reactions to A. invadans and cross-reacted with other Aphanomyces spp, though they may be useful for future studies on A. invadans. The other, designated MAb 3gJC9, only cross-reacted with the crayfish plague pathogen, A. astaci, and was used for the development of an immunohistochemistry protocol that may be of use in diagnosis. Immunohistochemistry with MAb 3gJC9, which recognised an extracellular product (ECP) of A. invadans, was specific to A. invadans in fish tissue, although it also recognised A. astaci in plague-infected crayfish. It also recognised the mycelium in fish infected with ulcerative mycosis, indicating that ulcerative mycosis is synonymous with EUS. Preliminary observations indicated that both ECPs and what appeared to be a hitherto unreported early stage of the mycelium are important in the pathology of EUS. Studies in vitro on the macrophages of EUS-susceptible giant gourami Osphronemus gouramy and silver barb Barbodes gonionotus, and EUS-resistant Nile tilapia Oreochromis niloticus, found that their macrophages were able to inhibit the growth of A. invadans. The macrophages of striped snakehead Channa striata did not inhibit A. invadans, which may account for their high EUS-susceptibility, especially as A. invadans strongly inhibited the respiratory burst of snakehead macrophages. Studies on humoral immune responses revealed that complement inhibited A. invadans in the case of snakeheads, gourami and barbs but not tilapia or swamp eels Monopterus albus. The humoral responses of the latter were very different to the four other species, and not elucidated. Low levels of anti A. invadans antibodies were found in tilapia and gourami from an EUS-endemic region, and high levels in snakehead. Snakehead antibodies appeared to be able to inhibit A. invadans even when complement was removed, but lower levels were produced at the low temperatures typically associated with EUS. A range of potential immunostimulants were screened for the ability to enhance resistance to EUS. The two successful products were administered as feed supplements to snakeheads and barbs that were subsequently injected intramuscularly with A. invadans. One, the algal extract Ergosan, showed some beneficial effects on snakeheads although the challenge was inconclusive. The other, the vitamin supplement Salar-bec, accelerated the cellular immune response and reduced mortality in snakeheads and barbs, and enhanced antibody production in snakeheads. The antibody response of snakeheads was further studied by comparing the anti- A. invadans antibody level, inhibitory activity of sera in vitro and protective capacity of sera from EUS-naïve snakeheads to that of snakeheads recently exposed to EUS and those subject to long term EUS-exposure. Sera of populations recently exposed to EUS showed an increased level of antibodies, but little improvement in inhibitory or protective activity. Sera from snakeheads that had endured long term exposure showed a wide range of antibody levels, but marked increases in inhibitory and protective activity. Antibodies cross-reacted with non-pathogenic Aphanomyces spp. in all cases.