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Clinical and Vaccine Immunology, October 2006, p. 1155-1161, Vol. 13, No. 10
1071-412X/06/$08.00+0     doi:10.1128/CVI.00058-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Comparison of the Proteosomes and Antigenicities of Secreted and Cellular Proteins Produced by Mycobacterium paratuberculosis

Donghee Cho and Michael T. Collins*

Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin—Madison, 2015 Linden Drive, Madison, Wisconsin 53706

Received 14 February 2006/ Returned for modification 14 April 2006/ Accepted 12 July 2006

The protein expression profiles and antigenicities of both culture filtrates (CF) and cellular extracts (CE) of Mycobacterium paratuberculosis were compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), one-dimensional electrophoresis (1-DE) and 2-DE immunoblotting, and enzyme-linked immunosorbent assay (ELISA). The CF proteins were harvested from supernatants of stationary-phase liquid cultures and concentrated by size exclusion filtration. The CE proteins were extracted by mechanical disruption of cells using glass beads and a high-speed agitator. Analysis of SDS-PAGE gels showed that the majority of CF proteins had low molecular masses (<50 kDa), whereas CE protein mass ranged more evenly over a broader range up to 100 kDa. By 2-DE, CF proteins had a narrow array of pI values, with most being between pH 4.0 and 5.5; CE proteins spanned pI values from pH 4.0 to 7.0. The antigenicities of CF and CE proteins were first determined by 1-DE and 2-DE immunoblotting with serum from a cow naturally infected with M. paratuberculosis. The serum reacted strongly to more proteins in the CF than the CE. Sera from 444 infected and 412 uninfected cattle were tested by ELISA with CF and CE as solid-phase antigens. Receiver-operator characteristic curve analysis of the ELISA results showed a significantly greater area under the curve for CF compared to CE (P < 0.05). A high degree of variability in protein binding patterns was shown with 1-DE immunoblot analysis with 31 sera from M. paratuberculosis-infected cattle. Collectively, these results indicate that serologic tests for bovine paratuberculosis may be improved by using proteins derived from CF instead of CE. To maximize the diagnostic sensitivity of serologic tests, multiple proteins will be required. Even so, a CF ELISA may not be able to detect all M. paratuberculosis-infected cattle, in particular those in the early stages of infection that have yet to mount an antibody response.

* Corresponding author. Mailing address: Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin—Madison, 2015 Linden Drive, Madison, WI 53706. Phone: (608) 262-8457. Fax: (608) 265-6463. E-mail: mcollin5{at}wisc.edu.

Clinical and Vaccine Immunology, October 2006, p. 1155-1161, Vol. 13, No. 10
1071-412X/06/$08.00+0     doi:10.1128/CVI.00058-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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