Enhancing the sensitivity of the agar gel immunodiffusion test for the diagnosis of bovine paratuberculosis through efficient media cultivation and modified loading pattern of surface antigens extracted by ethanol and vortex.

This study evaluated the efficacy of four different culture media in cultivating Mycobacterium avium subspecies paratuberculosis (MAP). Additionally, it examined the extraction of surface antigens through the suspension of harvested bacteria in ethanol, followed by vortex agitation. The potential of the extracted antigens in the agar gel immunodiffusion test (AGID) was evaluated by altering the loading pattern. This modification involved adding varying concentrations of antigens to peripheral wells and comparing the results to the conventional technique, which typically places a single antigen concentration in the center with sera surrounding it. The findings indicated that the Reid medium promoted the highest bacterial growth in the shortest period, yielding a larger volume of extractable antigens. Middlebrook 7H9 Glycerol Dextrose and Dorset Henley media ranked second and third, respectively, while Middlebrook 7H9-OADC required mycobactin enrichment and exhibited discrete bacterial growth foci, yielding minimal extracted antigens. The ethanol vortex-agar gel immunodiffusion (EV-AGID) demonstrated 92.3 % sensitivity and 100 % specificity in diagnosing bovine clinical paratuberculosis. The study detected 24 positive cases of Johne's disease in 26 Holstein-Friesian cows exhibiting clinical signs and isolated MAP bacteria from the fecal or intestinal mucosa. Furthermore, EV-AGID confirmed that 26 clinically healthy calves, which tested negative through ELISA and fecal cultures, were deemed disease-free. The modification of using serial dilutions of antigens increased sensitivity by 15.3 %. In conclusion, the findings affirm that a sensitive and specific immunodiffusion test can effectively confirm the diagnosis of bovine clinical paratuberculosis using a serial dilution of subspecies-specific surface antigens extracted via the ethanol and vortex technique.