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Due to the variability and possible absence of outward signs of dourine, clinical diagnosis is not always possible and laboratory diagnosis is necessary to confirm diagnoses of dourine.

The complement fixation test (CFT) is the prescribed test for international trade, and has been used successfully in eradication programs. Some uninfected animals, particularly donkeys, often have non-specific CFT reactions due to anticomplementary activity of their serum, thereby rendering results difficult to interpret. Indirect fluorescent antibody tests (IFAT) may help to resolve these cases.

Enzyme linked immunosorbent assays (ELISAs) and agar gel immunodiffusion (AGID) tests have also been used to diagnose dourine. Although no serological test is specific for dourine as cross-reactions occur with other trypanosomes (especially T. brucei and T. evansi), this is not a problem where these infections are all considered to be exotic and requiring eradication. CFT should always be used to test horses with clinical signs, to test horses that have been in contact with others who have or are at risk of having dourine and for official export certification. In such cases, samples for dourine (CFT) blood testing must be sent to the APHA, Weybridge (Tel: 01932 357335).

Definitive diagnosis by identification of the parasite is not undertaken for routine screening as the organisms are extremely difficult to find and are usually not detectable in blood smears. T. equiperdum cannot be distinguished microscopically from T. evansi.