APMIS zyxwvutsrq 99: 83-92, zyxwvutsrq 1991 Crossed immunoelectrophoretic analysis of Mycobacterium paratuberculosis MICHAEL T. COLLINS’, NIELS H0IBY2, J. BERG J0RGENSEN3, HERVE BERCOVIER4, RANDALL S. LAMBRECHTS AND ELLEN J0RGENSEN2 ‘Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin-Madison, Madi- son, Wisconsin, USA, *Statens Seruminstitut, Department of Clinical Microbiology at Rigshospitalet, Copenhagen, Denmark, 3National Veterinary Laboratory, Copenhagen, Denmark, 4The Hebrew University, Department of Clini- cal Microbiology at Hadassah Medical School, Jerusalem, Israel, and 5Dept. Health Sciences, University of Wiscon- sin-Milwaukee, Milwaukee, Wisconsin, USA Collins, M.T., Hniby, N., Jsrgensen, J.B., Bercovitr, H., Lambrecht, R.S. zyxw & Jnrgensen, E. Crossed immuno- electrophoretic analysis of Mycobacteriumparatuberculosis. APM IS 99: 83-92, I99 I. Antigenic analysis of M. paratuberculosis revealed extensive cross-reactivity with M. avium; however, the number of cross-reactive antigens found was dependent on the strain of M. avium tested. One antigen was shown to be the common antigen while another appeared to be iron-rgulated in its production. A commercial polyclonal antibody to M. paratuberculosis produced a CIE precipitin pattern comparable to that of the anti- body produced for the present study. zyxwvut An antigen designated no. 6 was consistently precipitated by sera from cattle infected with M. paratuberculosis. This antigen exhibited complete cross-reaction with M. avium and partial cross-reaction with M. phlei. Among three commercially available complement fixation (CF) antigen preparations widely used for serodiagnosis of Johne’s disease, none were shown to contain protein antigens that could be precipitated by M. paratuberculosis antibodies. A commercial antigen for use in an agar gel im- munodiffusion test for Johne’s disease diagnosis produced 12 precipitins with the M. paratuberculosis anti- body, one of which was identical with antigen 6. Key words: Mycobacterium paratuberculosis; crossed immunoelectrophoresis; serology; Johne’s disease; pa- ratuberculosis. M.T. Collins, Department of Pathobiological Sciences, University of Wisconsin-Madison, 2015 Linden Drive, Madison, Wisconsin, 53706 USA. Mycobacterium paratuberculosis causes disease principally in ruminants although it has also been isolated from human patients with Crohn’s disease (4, 5, 11). Prevalence of this highly infectious dis- ease appears to be increasing. Disease control ef- forts have been hampered by lack of a rapid, accur- ate and cost-effective diagnostic test. Efforts to de- vise a species-specific serological test by using ab- sorption techniques to increase test specificity have improved test accuracy significantly(16,17,24-26); however, this has comprised test sensitivity. A possi- Received April 10, 1990. Accepted July zyxwvutsr 24, 1990. ble solution to the problem might be use of a purified and concentrated species-specific antigen. From earlier studies on iron acquisition by Myco- bacterium paratuberculosis ( 12, 13), we hypothes- ized that this organism might produce iron-regulat- ed (IR) antigens. Such antigens could be unique to M. paratuberculosis and provide a new avenue for development of species-specific immunodiagnostic assays for Johne’s disease (paratuberculosis). Thus we initiated the investigation of M. paratuberculosis antigens herein described to test for the production of IR antigens, assess the seroreactivity of cattle to whole cell homogenates and search for the species- specificatigens reported in the earlier work of Gun- narsson & Fohtad (7). 83