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Clinical and Vaccine Immunology, May 2006, p. 535-540, Vol. 13, No. 5
1071-412X/06/$08.00+0     doi:10.1128/CVI.13.5.535-540.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

A Novel Enzyme-Linked Immunosorbent Assay for Diagnosis of Mycobacterium avium subsp. paratuberculosis Infections (Johne's Disease) in Cattle

Center for Wildlife Health, Department of Forestry, Wildlife and Fisheries, the University of Tennessee, Knoxville, Tennessee 37996,¹ Bacterial Diseases of Livestock Research Unit, National Animal Disease Center, Agricultural Research Service, United States Department of Agriculture, Ames, Iowa,² Immune System Section, Department of Immunology, National Institute of Animal Health, 3-1-5 Kannondai, Tsukuba, Ibaraki, Japan 305-0856,³ New Bolton Center, University of Pennsylvania, Kennett Square, Pennsylvania 19348-1692⁴

Received 13 January 2006/ Returned for modification 7 February 2006/ Accepted 9 March 2006

Enzyme-linked immunosorbent assays (ELISAs) for the diagnosis of Johne's disease (JD), caused by Mycobacterium avium subsp. paratuberculosis, were developed using whole bacilli treated with formaldehyde (called WELISA) or surface antigens obtained by treatment of M. avium subsp. paratuberculosis bacilli with formaldehyde and then brief sonication (called SELISA). ELISA plates were coated with either whole bacilli or sonicated antigens and tested for reactivity against serum obtained from JD-positive and JD-negative cattle or from calves experimentally inoculated with M. avium subsp. paratuberculosis, Mycobacterium avium subsp. avium, or Mycobacterium bovis. Because the initial results obtained from the WELISA and SELISA were similar, most of the subsequent experiments reported herein were performed using the SELISA method. To optimize the SELISA test, various concentrations (3.7 to 37%) of formaldehyde and intervals of sonication (2 to 300 s) were tested. With an increase in formaldehyde concentration and a decreased interval of sonication, there was a concomitant decrease in nonspecific binding by the SELISA. SELISAs prepared by treating M. avium subsp. paratuberculosis with 37% formaldehyde and then a 2-s burst of sonication produced the greatest difference (7x) between M. avium subsp. paratuberculosis-negative and M. avium subsp. paratuberculosis-positive serum samples. The diagnostic sensitivity and specificity for JD by the SELISA were greater than 95%. The SELISA showed subspecies-specific detection of M. avium subsp. paratuberculosis infections in calves experimentally inoculated with M. avium subsp. paratuberculosis or other mycobacteria. Based on diagnostic sensitivity and specificity, the SELISA appears superior to the commercial ELISAs routinely used for the diagnosis of JD.

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