Interpretation of the gamma interferon test for diagnosis of subclinical paratuberculosis in cattle

Abstract

A group of 252 cattle without clinical signs of paratuberculosis (paraTB) in 10 herds infected with paraTB and a group of 117 cattle in 5 herds without paraTB were selected. Whole-blood samples were stimulated with bovine, avian, and johnin purified protein derivative (PPD) and examined for gamma interferon (IFN-gamma) release. For diagnosis of paraTB, satisfactory estimated specificities (95 to 99%) could be obtained by johnin PPD stimulation irrespective of interpretation relative to bovine PPD or no-antigen stimulation alone, but numbers of test positives in the infected herds varied from 64 to 112 with different interpretation criteria. For a limited number of test-positive animals, no change in the test results could be observed with increasing antigen concentrations but IFN-gamma responses were significantly reduced (P < 0.0001) and four out of seven reactors tested negative when stimulation was performed on day-old samples. Denmark is free of bovine tuberculosis, but cross-reactivity with paraTB could be documented for cattle more than 14 months old in paraTB-infected herds compared with those in non-paraTB-infected herds. In both paraTB-free and paraTB-infected herds, false positives were observed when the test was applied to calves less than 15 months of age. Until novel antigen formulations more specific for these diseases are available, interpretation of the IFN-gamma test must be individually adjusted to fit specific needs and the context within which the test is applied and, for paraTB, the test seems most appropriate for use as a supportive tool for evaluation of disease-preventive measures in young stock.

FIG. 1.

Relationship between SEB or ConA as positive control stimulators and M. avium subsp. paratuberculosis PPDj as antigen-specific stimulation. OD readings were calibrated (cal.) with the positive and negative kit controls.

FIG. 2.

Lack of agreement between IFN-γ induction by M. avium subsp. avium PPDa or PPDj as shown by the difference in IFN-γ levels plotted against the average value following logarithmic transformation of OD values. SD, standard deviation.

FIG. 3.

Individual IFN-γ responses in herds infected or not infected with paraTB following whole-blood stimulation with PPDj, PPDa, or PPDb. Responses are presented as OD_C responses following specific stimulation minus no-antigen stimulation. Open circles indicate PPDj > PPDb in the top panel or PPDa > PPDb in the lower two panels. Closed circles indicate PPDb responses greater than or equal to the PPDj or PPDa response, respectively. The broken horizontal line at OD_C = 0.1 indicates the standard cutoff level at test interpretation for bovine paraTB or TB (bottom panel). The solid horizontal lines at 0.254 and 0.453 indicate the estimated 95% paraTB cutoff points for interpretation of PPDj or PPDa responses, respectively. Only IFN-γ OD_C levels of greater than 0.05 are shown; thus, e.g., results for 80 and 93 PPDj-stimulated animals in the infected and noninfected herds, respectively, are not shown (Table 2).