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United States Department of Agriculture

Agricultural Research Service

Research Project: IMMUNOLOGY AND INTERVENTION STRATEGIES FOR JOHNE'S DISEASE Title: Optimization of serum EVELISA for milk testing of Johne's disease

Authors
item Wadhwa, Ashutosh -
item Bannantine, John
item Byrem, Todd -
item Stein, Traci -
item Saxton, Arnold -
item Speer, C -
item Eda, S -

Submitted to: Foodborne Pathogens and Disease
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: May 2, 2012
Publication Date: August 1, 2012
Citation: Wadhwa, A., Bannantine, J.P., Byrem, T., Stein, T.L., Saxton, A.M., Speer, C.A., Eda, S. 2012. Optimization of serum EVELISA for milk testing of Johne's disease. Foodborne Pathogens and Disease. 9(8):749-754.

Interpretive Summary: Johne’s disease (JD) is caused by Mycobacterium avium subsp. paratuberculosis (MAP) and is one of the most widespread and economically important diseases of livestock and wild ruminants. Control of JD could be achieved by good herd management practices and diagnosis, but this approach has been hampered by the fact that the currently available commercial diagnostic tests suffer low sensitivity. In this study we optimized a previously developed serum ELISA to be used for testing milk samples. Milk samples, like serum samples, also contain antibodies to MAP, if the cow is infected. The results of this study demonstrate that anti-MAP antibodies are present in milk samples and can be detected in infected cows. This finding is of use to veterinarians and animal producers because milk samples are much easier to obtain than blood (serum) samples.

Technical Abstract: Johne’s disease (JD) or paratuberculosis, caused by Mycobacterium avium ssp. paratuberculosis (MAP), is one of the most widespread and economically important diseases of livestock and wild ruminants. Control of JD could be achieved by good herd management practices and diagnosis; however, this approach has been hampered by the fact that the currently available commercial diagnostic tests suffer low sensitivity. In our previous study, we developed a sensitive serum ELISA test, named EV (ethanol-vortex) ELISA using surface antigens of MAP extracted by a brief agitation of the bacteria in 80% ethanol using a vortex mixer. The objective of this study is to demonstrate that the EVELISA can be used for detection of anti–MAP antibodies in milk samples. In this study, we tested and optimized concentrations of antigen, milk, and secondary antibody for better differentiation of milk samples of cattle with MAP infections from those of cattle in JD–free herds. We evaluated five environmental mycobacteria as absorbents of cross reactive antibodies in milk and found that the mycobacteria had no significant effect on EVELISA results. Using the optimized conditions, a total of 57 milk samples from Holstein dairy cattle (37 animals found positive on the fecal PCR test and 20 animals from JD-free herds) were tested for anti-MAP antibody in milk by using the EVELISA method. The average of ELISA values in the JD-positive milk samples (mean ± SD = 0.355 ± 0.455) was significantly higher than that in the JD-negative milk samples (mean ± SD = 0.071 ± 0.011). These results warrant further studies for evaluation and validation of the EVELISA for milk testing of cattle for JD.

Last Modified: 12/24/2014
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