This project will develop methods and provide information for vaccine and novel treatment development. For example, techniques for the isolation and maintenance of N. pemaquidensis are based on monoxenic cultures. This culture is highly problematic because preparations of protozoa are contaminated with bacteria. Studies to determine cell function, protein and DNA composition have been seriously compromised by the bacteria. Culture relies on the use of agar. Cell propagation and harvesting by this system is time consuming and inefficient. Development of practical systems for cell factory production of N. pemaquidensis is required. This is important for studies of cell wall composition and cell function, which require considerable biomass. There is no model of infection using protozoa derived from monoxenic or xenic cultures. This represents a major limitation, particularly when it is necessary to use controlled doses of a single strain. Current methods rely on the use of N. pemaquidensis harvested from infected fish. While this strategy meets an immediate need, long-term it cannot be justified. Development of a method to grow in vitro virulent protozoa capable of infecting fish is an essential objective. The current library of N. pemaquidensis isolates obtained from fish with AGD is small and in continuous culture for almost 10 years. There is an urgent need to re-isolate N. pemaquidensis and expand the library to ensure an adequate range of phenotypes and genotypes. Preservation of N. pemaquidensis is an essential requirement of the AGD programme as it will maintain strain integrity, a vital objective for vaccine development. The complexity of growing N. pemaquidensis has proved a major limitation to studies on AGD. A centre of expertise in the culture of N. pemaquidensis should result in guaranteed supply of organism. A reference laboratory will ensure standardisation of cultures and uniformity of research outcomes.