Objective

Identification and characterization of novel antigens of M. paratuberculosis (M. ptb.) for vaccine and diagnostics development

Materials and methods

Genomic expression libraries were constructed using the λTripleEx™ (Clontech) and Zap-express® (Stratagene) expression vectors using genomic DNA from 4 week old liquid cultures of M. ptb. strains B854 and 316F, respectively. The genomic libraries were shown to represent >99.9% of the M. ptb. genome. Immunoscreening of phage plaques was carried out using serum from a cow naturally infected with M. ptb. Several positive phage recombinants were sequenced and potential antigens, encoded by these phages were expressed and purified as poly-histidine tagged proteins using dedicated expression vectors as pQE( Qiagen) or pET (Novagen). The immunological significance of these proteins was further analyzed in immunoblots and ELISAs using a serum collection containing sera from naturally and experimentally infected cattle.

Results

Among other antigens three novel secreted antigens were of 9 kDa, 15 kDa and 34 kDa were characterized, which display a strong homology to previously described M. tuberculosis antigens, TB 8.4, MPT53 and Erp, respectively. Using an arbitrary cut-off value of OD>0.3 at a serum dilution of >1/80, 11 out of 11 (100%) of serum samples from naturally infected and clinical stage cattle reacted with the novel 9 and 15 kDa novel antigens, and 10/11 (91%) with the novel 34 kDa antigen.