Open Access Highly Accessed Methodology article

Evaluation of ethanol vortex ELISA for detection of bovine tuberculosis in cattle and deer

Ashutosh Wadhwa1, Rachel E Johonson1, Keiko Eda1, W Ray Waters2, Mitchell V Palmer2, John P Bannantine2 and Shigetoshi Eda1*

Author Affiliations

1 Center for Wildlife Health, Department of Forestry, Wildlife and Fisheries, University of Tennessee Institute of Agriculture, 274 Ellington Plant Science Building, Knoxville, TN 37996, USA

2 Infectious Bacterial Diseases of Livestock Research Unit, National Animal Disease Center, USDA Agricultural Research Service, Ames, IA 50010, USA

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BMC Veterinary Research 2014, 10:147  doi:10.1186/1746-6148-10-147

Published: 4 July 2014



The use of serological assays for diagnosis of bovine tuberculosis (TB) has been intensively studied and use of specific antigens have aided in improving the diagnostic accuracy of the assays. In the present study, we report an in-house enzyme linked immunosorbent assay (ELISA), developed by using ethanol extract of Mycobacterium bovis (M. bovis). The assay, named (ethanol vortex ELISA [EVELISA]), was evaluated for detection of anti- M. bovis antibodies in the sera of cattle and white-tailed deer.


By using the EVELISA, we tested sera obtained from two species of animals; cattle (n = 62 [uninfected, n = 40; naturally infected, n = 22]) and white-tailed deer (n = 41 [uninfected, n = 25; naturally infected, n = 7; experimentally infected, n = 9]). To detect species specific molecules, components in the ethanol extract were analyzed by thin layer chromatography and western blotting.


Among the tested animals, 77.2% of infected cattle and 87.5% of infected deer tested positive for anti- M. bovis antibody. There were only minor false positive reactions (7.5% in cattle and 0% in deer) in uninfected animals. M. bovis -specific lipids and protein (MPB83) in the ethanol extract were detected by thin layer chromatography and western blotting, respectively.


The results warrant further evaluation and validation of EVELISA for bovine TB diagnosis of traditional and alternative livestock as well as for free-ranging animal species.

Bovine tuberculosis; Cattle; White-tailed deer; ELISA; Mycobacterium bovis; EVELISA