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In Australia, one of the biosecurity recommendations to help prevent the introduction of Mycoplasma bovis into a dairy herd is to use a PCR assay on bulk tank milk (BTM) samples to evaluate the M. bovis infection status of potential source herds. An alternative approach is to assess the immunological status of the herd with respect to previous exposure to M. bovis via the use of an ELISA that is commercially available for use on cattle milk and serum. The objectives of this study were to (1) evaluate factors potentially associated with variation in the ELISA BTM optical density coefficient (ODC%) in previously exposed herds, (2) evaluate the association between the proportion of cows that are ELISA positive and the BTM ELISA ODC%, (3) assess agreement between the BTM ELISA and PCR and culture, and (4) compare BTM ELISA ODC% between the “hospital” herd and the main lactating herd on the same farm. Bulk …