PCR is a highly accurate technique for confirming the presence of Mycobacterium avium subsp. paratuberculosis (Map) in broth culture. In this study, a simple, efficient, and low-cost method of harvesting DNA from Map cultured in liquid medium was developed. The proposed protocol (Universidad Austral de Chile [UACH]) was evaluated by comparing its performance to that of two traditional techniques (a QIAamp DNA Stool Mini Kit and cethyltrimethylammonium bromide [CTAB] method). The results were statistically assessed by agreement analysis for which differences in the number of cycles to positive (CP) were compared by Student's t-test for paired samples and regression analysis. Twelve out of 104 fecal pools cultured were positive. The final PCR results for 11 samples analyzed with the QIAamp and UACH methods or ones examined with the QIAamp and CTAB methods were in agreement. Complete (100%) agreement was observed between data from the CTAB and UACH methods. CP values for the UACH and CTAB techniques were not significantly different, while the UACH method yielded significantly lower CP values compared to the QIAamp kit. The proposed extraction method combines reliability and efficiency with simplicity and lower cost.
Animals ; Bacteriological Techniques/economics/*veterinary ; Cattle ; Cattle Diseases/diagnosis/*microbiology ; DNA, Bacterial/chemistry/genetics ; Female ; Mycobacterium avium subsp. paratuberculosis/*genetics ; Paratuberculosis/diagnosis/*microbiology ; Real-Time Polymerase Chain Reaction/veterinary ; Reproducibility of Results

Eighty-five complex (85A, 85B and 85C), 35-kDa and superoxide dismutase (SOD) were cloned, expressed and purified as antigens in an enzyme-linked immunosorbent assay (ELISA) to compare the serological reactivity of cows with different shedding levels of Mycobacterium avium subsp. paratuberculosis (MPT). Antibody responses to all recombinant antigens positively increased depending on shedding levels. In particular, antibody responses to the 35 kDa were higher than those to the others in all shedder groups. Also, the mean of O. D. values among Ag 85 complex, 85B showed slightly higher response than others with high sensitivity and specificity in all shedder groups. In receiver operating characteristic (ROC) curve analysis, the result of 35 kDa ELISA yielded an area under the curve value of 0.945 (95% confidence interval = 0.895 . 0.996), which indicated that this 35 kDa is more accurate indicator of MPT infection than other antigens. At the cut-off point recommended by the ROC curve analysis, the sensitivity and specificity of 35 kDa ELISA were higher than those of other antigens with 93.3% and 86.4%, respectively. Finally, a commercially available ELISA kit was used to clarify 200 positive and 200 negative sera. We then re-tested these serum samples with our ELISA test using the 35-kDa antigens. 35 kDa ELISA and commercial kit showed almost similar results in ROC curve analysis even though two of positive sera in commercial kit were negative in 35 kDa ELISA. The sera, which showed difference in the comparison with commercial ELISA kit, they also did not react with 35 kDa in Western blot. These results suggest that a 35-kDa based ELISA can be useful for detecting MPT infection.
Animals ; Antibodies, Bacterial/*immunology ; Antibody Formation/immunology ; Antibody Specificity/immunology ; Antigens, Bacterial/*immunology ; Blotting, Western ; Cattle ; Cloning, Molecular ; Electrophoresis, Polyacrylamide Gel ; Enzyme-Linked Immunosorbent Assay/veterinary ; Molecular Weight ; Mycobacterium paratuberculosis/*immunology ; Paratuberculosis/*diagnosis/microbiology ; Protein Biosynthesis ; Recombinant Fusion Proteins/*immunology ; Serologic Tests