title: Establishment of an antibody avidity test to differentiate vaccinated cattle from those naturally infected with Mycoplasma bovis.
abstract: Mycoplasma bovis is a major pathogen of bovine respiratory disease (BRD) in China and a live attenuated vaccine has recently been developed. This study aimed to establish an IgG avidity test to differentiate between naturally infected and vaccinated animals. An indirect ELISA (iELISA) was first established in the laboratory to detect antibodies specific to M. bovis using whole cell proteins as coating antigens and serum samples from experimentally infected cattle. The specificity and sensitivity of the iELISA was confirmed using a commercial ELISA kit as a reference standard. Both tests showed substantial agreement as indicated by a κ value of 0.78 (95% confidence interval, CI, 0.62, 0.93), and an overall 92.0% (80/87) agreement between the two tests. Based on the laboratory iELISA, a sodium thiocyanate (NaSCN) competitive iELISA was then developed for the detection of IgG avidity, expressed as relative avidity index (AI). Two-hundred and one experimentally immunised and naturally infected animals were used. These comprised 36 immunised calves, 38 negative control calves, 37 naturally infected calves, 87 calves of unknown status, and an additional three immunised calves that were used for a time trial. By testing true positive and negative antisera from either naturally infected or immunised calves, the AI cut-off value was defined as 70.4%. The diagnostic accuracy of the in-house NaSCN competitive iELISA was determined using serum samples collected from the experimental animals. The IgG avidity test demonstrated 96.0% sensitivity (95% CI 80.5%, 99.3%) and 95.8% specificity (95% CI 79.8%, 99.3%), and was successfully established as a valuable first test for differentiating vaccinated animals from those infected with M. bovis. This test may be a useful tool for clarifying the magnitude of M. bovis infection and in assessing the efficacy of vaccination in exposed animal populations.
authors.affiliation: The State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, China; College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China.
authors.forename: Xiaoxiao
authors.initials: X
authors.lastname: Han
authors.affiliation: The State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, China; College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China.
authors.forename: Farhan Anwar
authors.initials: FA
authors.lastname: Khan
authors.affiliation: The State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, China; College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China.
authors.forename: Xifang
authors.initials: X
authors.lastname: Zhu
authors.affiliation: The State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, China; College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China.
authors.forename: Rui
authors.initials: R
authors.lastname: Zhang
authors.affiliation: The State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, China; College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China.
authors.forename: Riaz
authors.initials: R
authors.lastname: Mustafa
authors.affiliation: The State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, China; College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China.
authors.forename: Changmin
authors.initials: C
authors.lastname: Hu
authors.affiliation: The State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, China; College of Animal Science, Huazhong Agricultural University, Wuhan 430070, China.
authors.forename: Yingyu
authors.initials: Y
authors.lastname: Chen
authors.affiliation: The State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, China; College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China; Key Laboratory of Development of Veterinary Diagnostic Products, Ministry of Agriculture, Wuhan 430070, China.
authors.forename: Huanchun
authors.initials: H
authors.lastname: Chen
authors.affiliation: The State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, China; College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China; Key Laboratory of Development of Veterinary Diagnostic Products, Ministry of Agriculture, Wuhan 430070, China. Electronic address: aizhen@mail.hzau.edu.cn.
authors.forename: Aizhen
authors.initials: A
authors.lastname: Guo
mesh_terms: D000818:Animals; D000907:Antibodies, Bacterial / Q000276:immunology*; D000915:Antibody Affinity / Q000276:immunology*; D001428:Bacterial Vaccines / Q000276:immunology*; D002417:Cattle; D002418:Cattle Diseases / Q000382:microbiology / Q000517:prevention & control*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods / Q000662:veterinary; D007074:Immunoglobulin G / Q000276:immunology; D009175:Mycoplasma Infections / Q000276:immunology / Q000517:prevention & control / Q000662:veterinary*; D045682:Mycoplasma bovis / Q000276:immunology*
chemical_list: D000907:Antibodies, Bacterial; D001428:Bacterial Vaccines; D007074:Immunoglobulin G
keywords: DIVA; IgG avidity test; Mycoplasma bovis; Natural infection; Vaccination