Development of a Recombinant Protein-Based Enzyme-Linked Immunosorbent Assay for Diagnosis of Mycoplasma bovis Infection in Cattle

• DOI: 10.1128/cvi.00670-13
• Publication Date: 2013-12-12T07:02:17Z
• Authors: Nadeeka K. Wawegama; Glenn F. Browning; Anna Kanci; Marc S. Marenda; Philip F. Markham
• Keywords: Mycoplasma bovis; Veterinary Medicine; 2. Zero hunger; Antigens, Bacterial; 0303 health sciences; Time Factors; Clinical Laboratory Techniques; Cattle Diseases; Enzyme-Linked Immunosorbent Assay; Antibodies, Bacterial; Sensitivity and Specificity; Recombinant Proteins; 3. Good health; 03 medical and health sciences; Immunoglobulin M; Immunoglobulin G; Animals; Cattle; Mycoplasma Infections

ABSTRACTMycoplasma boviscauses a range of diseases in cattle, including mastitis, arthritis, and pneumonia. However, accurate serological diagnosis of infection remains problematic. The studies described here aimed to identify an antigen that might be used to develop a more specific and sensitive diagnostic assay. A 226-kDa immunogenic protein was consistently detected in Western blots by antibodies in sera from calves experimentally infected withM. bovis. This protein was shown to be a membrane protein with lipase activity and was named mycoplasma immunogenic lipase A (MilA). Different regions of MilA were expressed inEscherichia colias glutathioneS-transferase (GST) fusion proteins and recombinant products from the amino-terminal end shown to have strong immunoreactivity withM. bovis-specific bovine sera. The most immunoreactive fusion protein, GST-MilA-ab, was used to develop indirect IgM and IgG enzyme-linked immunosorbent assays (ELISAs). The IgM ELISA detectedM. bovis-specific IgM antibody 2 weeks after infection with 97.1% sensitivity and had a specificity of 63.3%, while the IgG ELISA detectedM. bovis-specific IgG 3 weeks after infection with 92.86% sensitivity and had a specificity of 98.7%, demonstrating that the IgG ELISA has potential for use as a sensitive and specific assay for detecting infection in cattle.