Experimental infection models and diagnosis of epizootic ulcerative syndrome in three-spot gourami (Trichogaster trichopterus) and rainbow trout (Oncorhynchus mykiss)

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