| dc.description.abstract | As tuberculosis might be a problem in the declining Asian elephant population but too little is known about the exact prevalence of this disease, researchers in Bangkok, Chiang Mai, Utrecht and Pretoria are currently busy developing blood tests for detecting tuberculosis in elephants. During this research project, I participated in the development of an IFNγ assay by working on the expression of glycosylated IFNγ from insect cells, to be used as a positive control in the development of the IFNγ assay. I also evaluated sensitivity, specificity, positive and negative predictive value of indirect (i)ELISA tests employing ESAT6/CFP10, CFP10 & MBP83 antigens, and the commercial TB STAT-PAK rapid test.
The expression of IFNγ has encountered quite some obstacles, but we succeeded in constructing a pMT/BiP/V5-HisA vector carrying an IFNγ gene insertion (as assessed by PCR product size). This plasmid has been transferred into Schneider 2 insect cells (Drosophila melanogaster), using the Drosophila Expression System (DES®, Invitrogen). A stable expression of IFNγ by these cells has not been established yet.
The comparison of the different diagnostic tests for detecting TB infection in elephants provided some interesting results. Compared to the 100% sensitivity and 97% specificity claimed by the producer of the TB STAT-PAK test, in this project a sensitivity of 80% and a specificity of 87.23% were found. The iELISA test employing the immunodominant antigens ESAT6 & CFP10, showed a sensitivity of 75% and a specificity of 97.82%, and a positive predictive value of 88.24% opposed to a positive predictive value of 57.14% of the TB STAT-PAK test. Negative predictive values of these tests were about the same. This ESAT6/CFP10 iELISA test might be an alternative test to the expensive TB STAT-PAK test or trunk wash culture when screening for tuberculosis. However, limitations such as low sample size and –variation, growing body of evidence for a low sensitivity of the trunk wash culture (considered as the gold standard in this experiment), suboptimal negative controls used in the iELISA procedure, and the use of near to expiry STAT-PAK tests, suggest that more research has to be done regarding the use of this ESAT6/CFP10 iELISA test. | |
