title: Optimization of serum EVELISA for milk testing of Johne's disease.
abstract: Johne's disease (JD) or paratuberculosis, caused by Mycobacterium avium ssp. paratuberculosis (MAP), is one of the most economically important diseases of dairy cattle. Control of JD could be achieved by good herd management practices, and diagnosis; however, this approach has been hampered by the low sensitivity of currently available enzyme-linked immunosorbent assay (ELISA) tests. In our previous study, we developed a sensitive serum ELISA test, ethanol-vortex enzyme-linked immunosorbent assay (EVELISA), using ethanol extract of MAP. The objective of this study is to demonstrate that the EVELISA can be used for detection of anti-MAP antibodies in milk samples. In this study, we tested and optimized concentrations of antigen, milk, and secondary antibody for better differentiation of milk samples of cattle with MAP infections from those of cattle in JD-free herds. We evaluated five environmental mycobacteria as absorbents of cross-reactive antibodies in milk and found that the mycobacteria had no significant effect on EVELISA results. Using the optimized conditions, a total of 57 milk samples from Holstein dairy cattle (37 animals found positive on the fecal polymerase chain reaction test and 20 animals from JD-free herds) were tested for anti-MAP antibody in milk by using the EVELISA method. The average of ELISA values in the JD-positive milk samples (mean±SD=0.355±0.455) was significantly higher than that in the JD-negative milk samples (mean±SD=0.071±0.011). These results warrant further studies for evaluation and validation of the EVELISA for milk testing of cattle for JD.
authors: Center for Wildlife Health, Department of Forestry, Wildlife, and Fisheries, University of Tennessee, Knoxville, Tennessee 37996, USA.
authors: Ashutosh
authors: A
authors: Wadhwa
authors: John P
authors: JP
authors: Bannantine
authors: Todd M
authors: TM
authors: Byrem
authors: Traci L
authors: TL
authors: Stein
authors: Arnold M
authors: AM
authors: Saxton
authors: C A
authors: CA
authors: Speer
authors: Shigetoshi
authors: S
authors: Eda
mesh_terms: D000818:Animals; D000907:Antibodies, Bacterial / Q000302:isolation & purification*; D002417:Cattle; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods / Q000662:veterinary*; D005243:Feces / Q000737:chemistry / Q000382:microbiology; D008892:Milk / Q000382:microbiology*; D016927:Mycobacterium avium subsp. paratuberculosis / Q000276:immunology / Q000302:isolation & purification* / Q000472:pathogenicity; D010283:Paratuberculosis / Q000175:diagnosis*; D016133:Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary
chemical_list: D000907:Antibodies, Bacterial