Spore@Zr⁴⁺ Microspheres For The Detection Of Positive Antibody Against MPB83 Of Mycobacterium Bovis

Bovine tuberculosis is a major infectious disease caused by the intracellular bacterium Mycobacterium bovis. This disease is classified as class B infectious diseases by the world organization for animal health（OIE）, and as a second class animal disease in our country. The development of rapid, cost effective, high performance, and high sensitive methods to analysis M. bovis infect events are highly desired.In this work, spores of Bacillus amyloliquefaciens were prepared as immuno-microspheres via the adsorption of Zr⁴⁺. The spore@Zr⁴⁺ microspheres were then used for the detection of positive antibody against MPB83 of Mycobacterium bovis in the serum. Under the optimal conditions, the calibration plot obtained for the standard positive serum was linear within the dilution range of 1:1,000–20,000. The limit of detection for the assay was 1:32,000（S/N = 3）, which was significantly lower than ELISA（1:5,120; S/N = 3）. Repetitive measurements using 1:4,000-fold-diluted standard positive serum yielded reproducible results with a relative standard deviation of 2.16%（n = 11）. Sera infected with the causative agents of various bovine diseases, such as Bovine herpes-virus1, Babesia bovis, Foot-and-Mouth Disease Virus, Brucella abortus, Eperythrozoon wenyoni, were analyzed. Similar to the standard negative serum of Bovine Tuberculosis, sera infected with any of these diseases presented weak fluorescence signals, indicating a high specificity of the assay for Bovine Tuberculosis. Additionally, 69 clinical serum samples have been analyzed by both this proposed method and ELISA（IDEXX M.bovis USA）. The efficiency, sensitivity and specificity were 86.95%, 71.43% and 97.50% respectively, relative to the ELISA results.