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

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

Use of ethanol extract of Mycobacterium bovis for detection of specific antibodies in sera of farmed red deer (Cervus elaphus) with bovine tuberculosis

Background: Bovine tuberculosis (bTB) in wildlife species poses a threat to domestic livestock in many situations. Control programs for bTB in livestock depend on testing and slaughtering the positive animals; however, the currently available diagnostic tests often have poor specificity. In our previous study, we developed a specific and sensitive enzyme linked immunosorbent assay (ELISA) for another mycobacterial disease – Johne’s disease, using surface antigens of Mycobacterium avium ssp. paratuberculosis (MAP) extracted by briefly agitating the bacilli in 80% ethanol solution. The ELISA test was named ethanol vortex ELISA (EVELISA). The objective of this study is to examine whether EVELISA technique could be used to specifically detect anti-Mycobacterium bovis (M. bovis) antibodies in the serum of M. bovis-infected farmed red deer (Cervus elaphus). We tested a total of 45 red deer serum samples, divided in 3 groups – uninfected animals (n = 15), experimentally infected with M. bovis (n = 15) and experimentally infected with MAP (n = 15). Results: The presence of anti-M. bovis antibodies was tested using an ethanol extract of M. bovis. Without absorption of anti-MAP cross reactive antibodies, it was found that 13 out of the 15 MAP-infected animals showed high antibody binding. Using heat killed MAP as an absorbent of cross reactive antibodies, anti-M. bovis antibodies were detected in 86.7% of M. bovis-infected animals with minor false positive results caused by MAP infection. Conclusions: The results from this study suggest that EVELISA may form a basis for a sensitive and specific test for the diagnosis of bTB in farmed red deer

Early antibody response against Mycobacterium avium subspecies paratuberculosis antigens in subclinical cattle

<p>Abstract</p> <p>Background</p> <p>Our laboratories have previously reported on the experimental infection of cattle with <it>Mycobacterium avium </it>subsp <it>paratuberculosis </it>(<it>M. paratuberculosis</it>) using an intratonsillar infection model. In addition, we have recently developed a partial protein array representing 92 <it>M. paratuberculosis </it>coding sequences. These combined tools have enabled a unique look at the temporal analysis of <it>M. paratuberculosis </it>antigens within the native host. The primary objective of this study was to identify <it>M. paratuberculosis </it>antigens detected by cattle early during infection. A secondary objective was to evaluate the humoral immune response in cattle during the initial year of infection.</p> <p>Results</p> <p>Sera from two experimentally infected cattle, taken pre-inoculation and at day 70, 194 and 321 post infection, identified dynamic antibody reactivity among antigens with some showing an increased response over time and others showing declining levels of reactivity over the same time period. A <it>M. paratuberculosis </it>specific protein, encoded by MAP0862, was strongly detected initially, but the antibody response became weaker with time. The most reactive protein was a putative surface antigen encoded by MAP1087. A second protein, MAP1204, implicated in virulence, was also strongly detected by day 70 in both cattle. Subsequent experiments showed that these two proteins were detected with sera from 5 of 9 naturally infected cattle in the subclinical stage of Johne's disease.</p> <p>Conclusion</p> <p>Collectively these results demonstrate that <it>M. paratuberculosis </it>proteins are detected by sera from experimentally infected cattle as early as 70 days after exposure. These data further suggest at least two antigens may be useful in the early diagnosis of <it>M. paratuberculosis </it>infections. Finally, the construction and use of a protein array in this pilot study has led to a novel approach for discovery of <it>M. paratuberculosis </it>antigens.</p

Use of ethanol extract of Mycobacterium bovis for detection of specific antibodies in sera of farmed red deer (Cervus elaphus) with bovine tuberculosis

Background: Bovine tuberculosis (bTB) in wildlife species poses a threat to domestic livestock in many situations. Control programs for bTB in livestock depend on testing and slaughtering the positive animals; however, the currently available diagnostic tests often have poor specificity. In our previous study, we developed a specific and sensitive enzyme linked immunosorbent assay (ELISA) for another mycobacterial disease – Johne’s disease, using surface antigens of Mycobacterium avium ssp. paratuberculosis (MAP) extracted by briefly agitating the bacilli in 80% ethanol solution. The ELISA test was named ethanol vortex ELISA (EVELISA). The objective of this study is to examine whether EVELISA technique could be used to specifically detect anti-Mycobacterium bovis (M. bovis) antibodies in the serum of M. bovis-infected farmed red deer (Cervus elaphus). We tested a total of 45 red deer serum samples, divided in 3 groups – uninfected animals (n = 15), experimentally infected with M. bovis (n = 15) and experimentally infected with MAP (n = 15). Results: The presence of anti-M. bovis antibodies was tested using an ethanol extract of M. bovis. Without absorption of anti-MAP cross reactive antibodies, it was found that 13 out of the 15 MAP-infected animals showed high antibody binding. Using heat killed MAP as an absorbent of cross reactive antibodies, anti-M. bovis antibodies were detected in 86.7% of M. bovis-infected animals with minor false positive results caused by MAP infection. Conclusions: The results from this study suggest that EVELISA may form a basis for a sensitive and specific test for the diagnosis of bTB in farmed red deer

Brown Dwarfs in Young Moving Groups from Pan-STARRS1. I. AB Doradus

Substellar members of young ($\lesssim$150 Myr) moving groups are valuable benchmarks to empirically define brown dwarf evolution with age and to study the low-mass end of the initial mass function. We have combined Pan-STARRS1 (PS1) proper motions with optical$-$IR photometry from PS1, 2MASS and $\textit{WISE}$ to search for substellar members of the AB Dor Moving Group within $\approx$50 pc and with spectral types of late-M to early-L, corresponding to masses down to $\approx$30 M$_{Jup}$ at the age of the group ($\approx$125 Myr). Including both photometry and proper motions allows us to better select candidates by excluding field dwarfs whose colors are similar to young AB~Dor Moving Group members. Our near-IR spectroscopy has identified six ultracool dwarfs (M6$-$L4; $\approx$30$-$100 M$_{Jup}$) with intermediate surface gravities (INT-G) as candidate members of the AB Dor Moving Group. We find another two candidate members with spectra showing hints of youth but consistent with field gravities. We also find four field brown dwarfs unassociated with the AB Dor Moving Group, three of which have INT-G gravity classification. While signatures of youth are present in the spectra of our $\approx$125 Myr objects, neither their $J-K$ nor $W1-W2$ colors are significantly redder than field dwarfs with the same spectral types, unlike younger ultracool dwarfs. We also determined PS1 parallaxes for eight of our candidates and one previously identified AB Dor Moving Group candidate. Although radial velocities (and parallaxes, for some) are still needed to fully assess membership, these new objects provide valuable insight into the spectral characteristics and evolution of young brown dwarfs.Comment: ApJ, accepte