Concern about M. paratuberculosis infection of non-domestic hoofstock in US zoos is increasing. The Johne's Testing Center has completed radiometric culture assays for up to a third of the 176 zoos accredited in the US. M. paratuberculosis has been isolated from samples submitted by 9.1% of these institutions. Three of these institutions have had a significant prevalence of the disease in multiple species. Molecular fingerprinting of isolates is underway to assess the ability of these strain(s) to infect many types of ruminants. Over the last two years, approximately 50% of the mycobacteria isolated from faecal samples have been non-pathogenic, primarily saprophytic, organisms. The remaining 50% have been determined by genetic probe to be M. paratuberculosis (38.3%) and M. avium (11.7%). For one institution, histopathological confirmation of infection could be made in only 22% of animals for whom the organism was isolated from ante-mortem faecal samples. In an attempt to better characterise these cases, and to develop a more rapid and less expensive means of surveillance in these populations, the Johne's Testing Center has begun an assessment of the usefulness of a protein G ELISA ( IDEXX ) as a multi-species diagnostic tool. Sera from non-domestic hoofstock (both from confirmed infected animals and from animals belonging to the same species at institutions with no history of Johne's disease) are being tested. To date the binding patterns across numerous species at multiple serum dilutions appear comparable, although the optical density/dilution curves shift up or down depending on the species in question. Results using purified immunoglobulins, statistical analysis of the differences among and between individuals within the same genus/species and across genus/species and a comparison of the ELISA with AGID will be made.