Immunoglobulin G1 Enzyme-Linked Immunosorbent Assay for Diagnosis of Johne's Disease in Red Deer ( Cervus elaphus )

This study was designed to develop a customized enzyme-linked immunosorbent assay (ELISA) for the serodiagnosis of Johne’s disease (JD) in farmed deer. Two antigens were selected on the basis of their superior diagnostic readouts: denatured purified protein derivative (PPDj) and undenatured protoplasmic antigen (PpAg). ELISA development was based on the antigen reactivity of the immunoglobulin G1 (IgG1) isotype, which is a highly specific marker for mycobacterial disease seroreactivity in deer. Sensitivity estimates and test parameters were established using 102 Mycobacterium paratuberculosis-infected animals from more than 10 deer herds, and specificity estimates were determined using 508 uninfected animals from 5 known disease-free herds. A receiver-operated characteristic analysis determined that at a cut point of 50 ELISA units, there was a specificity of 99.5% and sensitivities of 84.0% with PPDj antigen, 88.0% with PpAg, and 91.0% when the antigens were used serially in a composite test. Estimated sensitivity was further improved using recombinant protein antigens unique for M. paratuberculosis, which identified infected animals that were unreactive to PPDj or PpAg. While 80% of animals that were seropositive in the IgG1 ELISA had detectable histopathology, the assay could also detect animals with subclinical disease. The test was significantly less sensitive (75%) for animals that were culture positive for M. paratuberculosis but with no detectable pathology than for those with pathological evidence of JD (>90%). When the IgG1 ELISA was used annually over a 4-year period in a deer herd with high levels of clinical JD, it eliminated clinical disease, increased production levels, and reduced JD-related mortality. Johne’s disease (JD) is a chronic enteritis found in ruminants, caused by infection with the bacterium Mycobacterium paratuberculosis, that produces major economic losses in livestock and dairy industries worldwide. Infection due to M. paratuberculosis manifests as a chronic inflammatory gastroenteritis, with epithelial thickening in the lower intestine causing malabsorption of nutrients and leading to wasting and eventual death in affected animals. In most ruminants, it can take several years for clinical symptoms of JD to present, highlighting the chronic nature of the disease. In deer, however, the process from infection to death can progress more rapidly, with animals dying from the disease as early as 8 months of age (21). This more acute presentation of pathology suggests that red deer (Cervus elaphus) may provide a more informative infection model to track immunological and etiological pathways of M. paratuberculosis infection. Outbreaks of JD have been reported in young deer (8 to 15 months), with death in 20% of animals; older animals can also sporadically present with clinical JD typical of that found in cattle and sheep, and this may be exacerbated by stress or aging (21). JD can be spread horizontally among adult animals and may also be spread pseudovertically during pregnancy. In some instances, viable