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

Clostridium (C.) difficile is a common cause of nosocomial diarrhea in horses. Vancomycin and metronidazole have been used as standard treatments but are only moderately effective, which highlights the need for a novel alternative therapy. In the current study, we prepared antiserum of equine origin against both C. difficile toxins A and B as well as whole-cell bacteria. The toxin-neutralizing activities of the antibodies were evaluated in vitro and the prophylactic effects of in vivo passive immunotherapy were demonstrated using a conventional mouse model. The data demonstrated that immunized horses generated antibodies against both toxins A and B that possessed toxin-neutralizing activity. Additionally, mice treated with the antiserum lost less weight without any sign of illness and regained weight back to a normal range more rapidly compared to the control group when challenged orally with 10(7) C. difficile spores 1 day after serum injection. These results indicate that intravenous delivery of hyperimmune serum can protect animals from C. difficile challenge in a dose-dependent manner. Hence, immunotherapy may be a promising prophylactic strategy for preventing C. difficile infection in horses.
Animals ; Antibodies, Bacterial/blood/*immunology/therapeutic use ; Bacterial Proteins/immunology/therapeutic use ; Bacterial Toxins/immunology/therapeutic use ; Clostridium Infections/microbiology/prevention & control/*veterinary ; Clostridium difficile/*immunology ; Enterotoxins/immunology/therapeutic use ; Female ; Horse Diseases/microbiology/*prevention & control ; Horses ; Immune Sera/*immunology ; Immunization, Passive/*veterinary ; Mice ; Mice, Inbred C57BL ; Spores, Bacterial/immunology