Failure to detect M. avium subspecies paratuberculosis in Johne's disease using a proprietary fluorescent in situ hybridization assay.

OBJECTIVES: Mycobacterium avium subspecies paratuberculosis (MAP) causes Johne's disease in ruminants. The "gold standard" of MAP detection is by culture, DNA sequencing possibly supplemented by identification of Ziehl-Neelsen positive mycobacteria. The purpose of this study was to evaluate a proprietary (Affymetrix™ RNA view®) fluorescent in situ hybridization (FISH) assay for MAP RNA. Intestine from a steer with documented Johne's disease was assayed according to the manufacturer's instructions. Probes were custom designed for MAP and bovine ß-actin (as the eukaryotic housekeeping gene) from published genomes. We attempt to prevent false positive signal in the "no-probe" control, by modifying wash solutions, using recommended hydrochloric acid titration and different fluorescent filters (TritC for Texas Red and "Hope" for Cy-5). RESULTS: Repetitively, false positive signal was observed in our "no probe" negative control. Attempts to correct this according to the manufacturers suggestions, and with multiple derivative techniques have been unsuccessful. It is concluded that when performed according to manufactures instruction and with multiple variations on the manufactures recommended suggestions to correct for false positive signal, that the Affymetrix™ RNA view® cannot be used to detect MAP in pre-frozen intestine of cattle with Johne's disease.

Identification of Novel Antigens Recognized by Serum Antibodies in Bovine Tuberculosis.

Bovine tuberculosis (TB), caused by Mycobacterium bovis, remains an important zoonotic disease posing a serious threat to livestock and wildlife. The current TB tests relying on cell-mediated and humoral immune responses in cattle have performance limitations. To identify new serodiagnostic markers of bovine TB, we screened a panel of 101 recombinant proteins, including 10 polyepitope fusions, by a multiantigen print immunoassay (MAPIA) with well-characterized serum samples serially collected from cattle with experimental or naturally acquired M. bovis infection. A novel set of 12 seroreactive antigens was established. Evaluation of selected proteins in the dual-path platform (DPP) assay showed that the highest diagnostic accuracy (∼95%) was achieved with a cocktail of five best-performing antigens, thus demonstrating the potential for development of an improved and more practical serodiagnostic test for bovine TB.

Inferring biomarkers for Mycobacterium avium subsp. paratuberculosis infection and disease progression in cattle using experimental data.

Available diagnostic assays for Mycobacterium avium subsp. paratuberculosis (MAP) have poor sensitivities and cannot detect early stages of infection, therefore, there is need to find new diagnostic markers for early infection detection and disease stages. We analyzed longitudinal IFN-γ, ELISA-antibody and fecal shedding experimental sensitivity scores for MAP infection detection and disease progression. We used both statistical methods and dynamic mathematical models to (i) evaluate the empirical assays (ii) infer and explain biological mechanisms that affect the time evolution of the biomarkers, and (iii) predict disease stages of 57 animals that were naturally infected with MAP. This analysis confirms that the fecal test is the best marker for disease progression and illustrates that Th1/Th2 (IFN-γ/ELISA antibodies) assays are important for infection detection, but cannot reliably predict persistent infections. Our results show that the theoretical simulated macrophage-based assay is a potential good diagnostic marker for MAP persistent infections and predictor of disease specific stages. We therefore recommend specifically designed experiments to test the use of a based assay in the diagnosis of MAP infections.