Diagnosis of Encephalitozoon cuniculi (ECUN) remains difficult despite advances in serological testing including the use of adjunct testing for C reactive protein in addition to quantitative IgM and IgG titers. After delineation of antigens targeted in the humoral immune response of rabbits to ECUN by combined Western blot and mass spectrometry methods, an ELISA using a recombinant antigen preparation of polar tube protein 2 (PTP2) was developed. This antigen is expressed during the actual infection of a new host cell. Notably, while significant correlations were observed for IgM (r=0.46, p=0.0003) and IgG (r=0.63, p<0.0001) detection versus the original fully antigen ELISA, these two methods are not equivalent. IgM reactivity also appears to be higher, perhaps reflecting the increased specificity found with the PTP2 antigen. Changes in titers versus clinical presentation and post treatment have also been examined. Concurrent with this continuing study of test specificity and sensitivity, a second study has been undertaken to examine changes of titers over time in three cohorts of rabbits: clinically normal, ECUN suspect, and non ECUN suspect but clinically abnormal rabbits. The goal is to gauge the variability of titers in the absence and presence of clinical signs. One notable observation to date is the identification of clinically normal rabbits as well as post treatment rabbits, many with incomplete resolution of signs, which have prolonged expression of anti-ECUN IgM antibody. A greater understanding of this observation may provide valuable information in the understanding of the pathophysiology of infection including recurrence of clinical signs.