A novel enzyme-linked immunosorbent assay for diagnosis of Mycobacterium avium subsp. paratuberculosis infections (Johne's Disease) in cattle

Abstract

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.

FIG. 1.

Effect of formaldehyde treatment on the ability of IgG in bovine serum to bind to whole organisms or to sonicated antigens of Mycobacterium avium subsp. paratuberculosis in an ELISA. The ELISA was conducted using whole cells (WELISA) (A) and sonicated antigens (SELISA) (B). M. avium subsp. paratuberculosis bacilli were treated with distilled water or 37% formaldehyde and not sonicated (A) or sonicated for 2 s and with the supernatant collected (B) immobilized via evaporation to the wells of 96-well plates. Immobilized M. avium subsp. paratuberculosis antigens were then treated with M. avium subsp. paratuberculosis-positive (solid bars) or -negative (cross-hatched bars) serum and then treated with biotinylated anti-bovine IgG polyclonal antibody. Controls (open bars) consisted of M. avium subsp. paratuberculosis treated with distilled water or formaldehyde and then secondary biotinylated antibody and HRP-streptavidin only. Each bar represents the mean + standard deviation of five replications. In both the WELISA and SELISA, treatment with formaldehyde minimized nonspecific antibody reactions. This experiment was repeated three times with similar results.

FIG. 2.

Effects of various formaldehyde concentrations on IgG binding to M. avium subsp. paratuberculosis antigens by SELISA. After formaldehyde treatment, whole M. avium subsp. paratuberculosis bacilli were sonicated for 2 s and the supernatant was used for the SELISA. Solid circles, pool of serum from five cattle that previously tested M. avium subsp. paratuberculosis positive by the FCM; open circles, a pool of serum from five cattle previously tested M. avium subsp. paratuberculosis negative by the FCM; open triangles, no serum. Each data point represents the mean of duplicate experiments. The greatest difference between M. avium subsp. paratuberculosis-positive and -negative serum occurred with the highest concentration of formaldehyde (37%) tested.

FIG. 3.

Effects of duration of sonication on IgG binding to M. avium subsp. paratuberculosis antigens by SELISA. M. avium subsp. paratuberculosis bacilli were treated with 37% formaldehyde and sonicated for various time intervals, and the supernatant was used in the SELISA. Solid circles, pool of serum from five cattle previously tested M. avium subsp. paratuberculosis positive by the FCM; open circles, pool of serum from five cattle previously tested as M. avium subsp. paratuberculosis negative by the FCM; open triangles, no serum. Each data point represents the mean of duplicate experiments. A 2-s sonication gave the greatest difference between M. avium subsp. paratuberculosis-positive and -negative serum.

FIG. 4.

Diagnostic sensitivity of SELISA. (Column 1) Serum samples from cattle on dairy farms that had tested JD negative for 5 consecutive years by ELISA and by fecal culture, PCR, and ELISA performed during the fifth year. (Column 2) Serum samples from dairy cattle that tested JD positive by fecal culture. A cutoff value of 0.23 was used to distinguish M. avium subsp. paratuberculosis negatives and positives. Similar results were obtained in two separate experiments. The SELISA showed no false positives and only one negative with M. avium subsp. paratuberculosis-positive serum. (Note that this negative might have been a false positive [pass-through] by the fecal culture test.)

FIG. 5.

Specific detection of M. avium subsp. paratuberculosis infections by SELISA in cattle experimentally inoculated with M. avium subsp. paratuberculosis, Mycobacterium avium subsp. avium, and M. bovis. Serum was obtained from calves at time of intratonsillar inoculation of mycobacteria (M. avium subsp. paratuberculosis, M. avium subsp. avium, or M. bovis [panels A to C, respectively]) and then at 1- or 2-week intervals thereafter for up to 320 days. The SELISA was used to test for reactivity against M. avium subsp. paratuberculosis. Each symbol indicates an individual animal. (A) SELISA of IgG binding to M. avium subsp. paratuberculosis antigens in serum from calves inoculated with M. avium subsp. paratuberculosis (n = 3; solid squares, 5903; solid diamonds, 5902; and solid triangles, 5904). (B) SELISA of IgG binding to M. avium subsp. paratuberculosis antigens by serum from calves inoculated with M. avium subsp. avium (n = 3; solid diamonds, 6137; solid triangles, 193; and solid squares, 2016). (C) SELISA of IgG binding to M. avium subsp. paratuberculosis in serum from calves inoculated with M. bovis (n = 2; squares, 202; and triangles, 2354). Similar results were obtained in two separate experiments. The positive fluorescence levels from day 0 to approximately day 100 in panels B and C are probably due to the presence of maternal antibodies against M. avium subsp. paratuberculosis. The SELISA detected M. avium subsp. paratuberculosis-specific antibodies at 174 days after inoculation (A) but did not cross-react with serum from M. avium subsp. avium- or M. bovis-inoculated calves (B and C).