title: The modification and evaluation of an ELISA test for the surveillance of Mycobacterium avium subsp. paratuberculosis infection in wild ruminants.
abstract: BACKGROUND
  Enzyme-linked immunosorbent assay (ELISA) is often used to test wildlife samples for Mycobacterium avium subsp. paratuberculosis (MAP) infection. However, commercially available kits are only validated for use with domestic ruminant species. A literature review was performed to document the current use of MAP serum ELISA in wild and semi-domestic ruminants. We then modified and evaluated a commercial ELISA kit (IDEXX Mycobacterium paratuberculosis Antibody Test Kit) for use with species for which it was not originally developed: elk (Cervus elaphus), bison (Bison bison) and caribou (Rangifer tarandus). We tested the affinity of different conjugates for immunoglobulin G (IgG) isolated from these species, performed checkerboard tests to determine the optimal dilutions of samples and conjugates, and established cut-off values using two different methods: a Receiver Operational Curve on a panel of known samples for elk, and an alternate method involving a panel of unknown serum samples for the three species.
  
  
  RESULTS
  We found that the anti-bovine conjugate included in the IDEXX ELISA kit has limited affinity for elk, bison, and caribou IgG. Protein G showed good affinity for IgG of all three species, while anti-deer conjugate also bound elk and caribou IgG. Using Protein G with elk serum, a cut-off sample-to-positive (S/P) value of 0.22 was selected, resulting in a sensitivity and specificity of 73% and 90%, respectively, whereas, using an anti-deer conjugate with elk serum, an S/P cut-off value of 0.29 gave a sensitivity of 68%, with 100% specificity. Cut-off values for bison and caribou using the Protein G conjugate were 0.17 and 0.25 respectively.
  
  
  CONCLUSIONS
  Due to incomplete reporting and a lack of test validation, it is difficult to critically appraise results of many sero-surveys that have previously been done for MAP in wildlife. Commercial ELISA kits may have limited or no capacity to detect antibodies from species other than for which they were developed. In order to generate reliable test results, it is essential to evaluate the test and perform modifications if deemed necessary. Despite the challenges inherent to wildlife diagnostics, we have shown that several methods can be used to improve confidence in test results.
authors.affiliation: Faculty of Veterinary Medicine, University of Calgary, 3330 Hospital Drive NW, Calgary, AB T2N 4N1, Canada. mpruvot@ucalgary.ca.
authors.forename: Mathieu
authors.initials: M
authors.lastname: Pruvot
authors.forename: Taya L
authors.initials: TL
authors.lastname: Forde
authors.forename: Jillian
authors.initials: J
authors.lastname: Steele
authors.forename: Susan J
authors.initials: SJ
authors.lastname: Kutz
authors.forename: Jeroen
authors.initials: J
authors.lastname: De Buck
authors.forename: Frank
authors.initials: F
authors.lastname: van der Meer
authors.forename: Karin
authors.initials: K
authors.lastname: Orsel
mesh_terms: D000818:Animals; D000835:Animals, Wild / Q000382:microbiology*; D016164:Bison / Q000382:microbiology*; D003670:Deer / Q000382:microbiology*; D004797:Enzyme-Linked Immunosorbent Assay / Q000662:veterinary*; D016927:Mycobacterium avium subsp. paratuberculosis; D010283:Paratuberculosis / Q000175:diagnosis*; D011159:Population Surveillance / Q000379:methods; D011933:Reagent Kits, Diagnostic / Q000662:veterinary; D012680:Sensitivity and Specificity
chemical_list: D011933:Reagent Kits, Diagnostic