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United States Department of Agriculture

Agricultural Research Service

Title: Time Delay, Temperature Effects and Assessment of Positive Controls on Whole Blood for the Gamma Interferon Elisa to Detect Paratuberculosis

item Robbe Austerman, Suelee
item Stabel, Judith

Submitted to: Journal of Veterinary Medicine
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: January 1, 2006
Publication Date: June 1, 2006
Citation: Robbe Austerman, S., Krull, A.C., Stabel, J.R., Palmquist, D.E. 2006. Time Delay, Temperature Effects and Assessment of Positive Controls on Whole Blood for the Gamma Interferon ELISA to Detect Paratuberculosis. American Journal of Veterinary Research. 53(5):213-217.

Interpretive Summary: Johne’s disease is an important disease in cattle, sheep and goats in the USA. Currently there are no approved diagnostic tests to detect animals in the early stages of infection. The gamma interferon test is a potential test that can be used in cattle. However, blood must be submitted to the lab while the blood cells are still active. This study looked at time delay and temperature effects on blood for the gamma interferon test. The findings were that whole blood should be stored/transported at ambient room temperature. Submission to the laboratory for best results should be submitted with in 8 hours. While most strong positive cows will remain positive after a 24 hour time delay, weak positive cows may be negative.

Technical Abstract: Our objectives were to evaluate the effects of time delay and temperature on whole blood used in the gamma interferon (IFN-gamma) ELISA for paratuberculosis, evaluate 4 potential positive controls, and to assess the potency of 4 different mycobacterial antigens in the IFN-gamma ELISA. Nine adult Holstein cattle naturally infected with Mycobacterium avium subspecies paratuberculosis with a previous history of positive IFN-gamma responses were used in the study. A randomized complete block design was used. Sixty blood tubes were collected from each animal and held at 48.9, 37.8, 26.7, 21.1, 15.6, and 4.4 (degrees C). Samples were removed from these temperatures at 9 different time points and then exposed to 4 different mycobacterial antigens and 4 potential positive controls. The plasma was then assayed with a commercial IFN-gamma ELISA. Blood stored at 21.1 and 15.6 (degrees C) maintained the highest INF-gamma activity with severe deleterious effects at or above 37.8 (degrees C). The rate of IFN-gamma production loss was similar between cows regardless of their initial ELISA OD value. The PPD's were more potent at stimulating IFN-gamma production than a whole cell sonicate. Considering positive controls, only phytohemagglutinin A somewhat mimicked the mycobacterial antigen response.

Last Modified: 4/19/2015