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Updated: Oct 7, 2026
Application of Long-term cultured Interferon-γ Enzyme-linked Immunospot Assay for Assessing Effector and Memory T Cell Responses in Cattle
Published on: July 11, 2015
An evaluation of a modified interferon-gamma assay for the detection of paratuberculosis in dairy herds
1Bacterial Diseases of Livestock Research Unit, National Animal Disease Center, 2300 Dayton Road, Ames, IA 50010, USA. jstabel@nadc.ars.usda.gov
Abstract:
Whole blood samples were obtained from multiple dairy herds in Pennsylvannia and in Wisconsin which were previously determined to be infected with Mycobacterium paratuberculosis (MpS) (Johne's disease) by fecal culture. Blood samples were shipped overnight to the National Animal Disease Center (NADC) in Ames, IA for processing and interferon-gamma (IFN-gamma) analysis. Blood samples were incubated alone (non-stimulated) or with concanavalin A (ConA), a T-cell mitogen used as a positive control in the assay, for 18h. In addition, samples were incubated with M. avium purified protein derivative (AvPPD), M. bovis purified protein derivative (BoPPD), or a whole cell sonicate of M. paratuberculosis for 18h to elicit antigen-specific IFN-gamma production. After incubation, plasma was harvested and analyzed for IFN-gamma by ELISA. Values for IFN-gamma for non-stimulated blood samples (background) were consistently low for animals in all herds evaluated. In contrast, ConA stimulation of blood samples evoked a significant secretion of IFN-gamma regardless of infection status or fecal culture results for individual cows, indicating that immune cells were still viable after overnight shipment and capable of responding to stimulation. Antigen-specific IFN-gamma results were positively correlated with infection status as determined by previous fecal shedding and/or current fecal shedding of M. paratuberculosis. Accuracy of the IFN-gamma assay for correctly predicting infection status of individual cows in the herds with low levels of infection ranged from 50 to 75% when used as a single test. Combined use of the IFN-gamma test and a commercial ELISA antibody test accurately predicted infection status of 73% of cows from a dairy herd with a high level of M. paratuberculosis infection and 90% from a well-characterized group of dairy cows at the NADC. These results indicate that the antigen-specific IFN-gamma assay is a very sensitive diagnostic tool for detection of subclinical paratuberculosis in cattle and may be useful on an individual animal basis to remove infected animals from the herd.
Insights
The interferon-gamma (IFN-gamma) assay shows promise for detecting Mycobacterium paratuberculosis (Johne's disease) in dairy cattle. Combining this test with antibody tests improves accuracy in identifying infected animals for herd management.
Area of Science:
- Veterinary Immunology
- Bovine Infectious Diseases
- Diagnostic Assays
Background:
- Mycobacterium paratuberculosis (MpS) causes Johne's disease in cattle, leading to significant economic losses in dairy herds.
- Accurate and early detection of MpS infection is crucial for effective herd management and disease control.
- Current diagnostic methods, such as fecal culture and antibody tests, have limitations in sensitivity, especially in early or subclinical stages of infection.
Purpose of the Study:
- To evaluate the diagnostic accuracy of an antigen-specific interferon-gamma (IFN-gamma) assay for detecting Mycobacterium paratuberculosis infection in dairy cattle.
- To assess the utility of the IFN-gamma assay as a standalone test and in combination with a commercial ELISA antibody test.
- To determine the potential of the IFN-gamma assay for identifying subclinically infected cattle.
Main Methods:
- Whole blood samples were collected from dairy herds infected with MpS.
- Blood samples were incubated with specific antigens (M. avium PPD, M. bovis PPD, M. paratuberculosis sonicate) or mitogen (ConA) to stimulate IFN-gamma production.
- Plasma IFN-gamma levels were measured using ELISA.
- Results were correlated with infection status determined by fecal culture and compared with a commercial ELISA antibody test.
Main Results:
- Antigen-specific IFN-gamma production was positively correlated with MpS infection status.
- The IFN-gamma assay alone achieved 50-75% accuracy in predicting infection in herds with low infection levels.
- Combining the IFN-gamma test with a commercial ELISA antibody test improved accuracy to 73% in a high-prevalence herd and 90% in a well-characterized group.
Conclusions:
- The antigen-specific IFN-gamma assay is a sensitive diagnostic tool for detecting subclinical paratuberculosis in cattle.
- This assay may be valuable for individual animal assessment to identify and remove infected animals from dairy herds.
- The combined use of IFN-gamma and antibody testing offers enhanced diagnostic accuracy for Johne's disease management.
