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

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, components in the ethanol extract were analyzed 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.

Figure 1

A - Ethanol-vortex enzyme linked immunosorbent assay (EVELISA). Antibody binding on serum samples from uninfected (n = 40) and naturally infected (n = 22) cattle were tested by EVELISA test. All the uninfected animals were from a bovine TB free herd and the naturally infected animals were determined as infected by mycobacterial culture, histology and IS6110 PCR techniques. A cut-off value of 0.4 was used to distinguish M. bovis negatives and positive animals. B - EVELISA results from sera of deer: Antibody binding in serum of uninfected (n = 25), M. bovis -infected – naturally (n = 7) and experimentally (n = 9) deer was determined using the EVELISA test. A cut-off value of 0.068 was used to distinguish between M. bovis -negative and positive animals. Both the cut-off values were determined to maximize the sum of sensitivity and specificity values. All experiments were conducted in duplicate or triplicate and repeated at least twice. The statistical difference of antibody binding was evaluated using Mann–Whitney U test.

Figure 2

Thin layer chromatography (TLC) (A) and immunoblot (B) of ethanol extracts of mycobacterial isolates. (A) Dried ethanol extracts of mycobacterial isolates were Folch washed and then developed on a silica-gel TLC plate using a mixture of Chloroform: Methanol: Water (90:10:1) to detect the presence of species specific molecules in the extract of M. bovis. The plates were sprayed with polymolybdate solution and the molecules were heated prior to visualization. Ethanol extracts of M. bovis, MAP (K10 Strain), MAP (Linda Strain) and M. avium subsp. avium were used in the lanes 1, 2, 3 and 4, respectively. (B) The MPB83 protein is present in the M. bovis ethanol extract. Proteins in M. bovis ethanol extract were separated by SDS-PAGE and transferred on to a nitrocellulose membrane. An anti-MPB83 monoclonal antibody labeled both the native MPB83 in the ethanol extract (lane 1, arrow) and the recombinant MPB83 fusion protein (lane 2, arrow).