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Title:   Author  item
                                            WADHWA, ASHUTOSH - University Of Tennesseeitem
                                            JOHNSON, RACHEL - University Of Tennesseeitem
                                            EDA, KEIKO - University Of Tennesseeitem
                                            Waters, Wadeitem
                                            Palmer, Mitchellitem
                                            Bannantine, Johnitem
                                            EDA, SHIGETOSHI - University Of Tennessee
  Submitted to: BioMed Central (BMC) Veterinary Research
                                    Publication Type: Peer Reviewed Journal
                                    Publication Acceptance Date: 12/6/2013
                                    Publication  Date: 7/4/2014
                                    Citation: Wadhwa, A., Johnson, R.E., Eda, K., Waters, W.R., Palmer, M.V., Bannantine, J.P., Eda, S. 2014. Evaluation of ethanol vortex ELISA for detection of bovine tuberculosis in cattle and deer. BioMed Central (BMC) Veterinary Research. 10(1):147.Interpretive Summary: Despite highly successful eradication efforts in several countries, tuberculosis of cattle remains a serious health concern worldwide. In addition, recent outbreaks of tuberculosis in Michigan, Minnesota, California, Washington, Texas, Nebraska, Indiana, Colorado, and New Mexico demonstrate that the disease is far from eliminated from the United States. Control of bovine tuberculosis is hindered by the presence of wildlife reservoirs, such as white-tailed deer in Michigan, continued importation of tuberculosis-infected cattle from Mexico, and failure of current testing strategies to detect infected animals. Improved techniques are needed to detect tuberculosis-infected cattle. In the present study, a simple blood-based test was developed to detect tuberculosis infected cattle or white-tailed deer. These findings demonstrate the feasibility for development of novel and simple tests to detect bovine tuberculosis. Knowledge obtained from this study will enable more accurate detection of cattle with tuberculosis; thereby, enhancing the tuberculosis eradication program.
                                Technical Abstract: Background
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.
  
Methods
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, dried ethanol extract was used by thin layer chromatography and western blotting.
   
Results
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.
 
Conclusion
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.


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