Proposal of an iELISA for Mycoplasma bovis diagnosis in dairy cattle and associated risk factors

ABSTRACT Mycoplasma bovis is a highly contagious agent associated with several pathologies in cattle. The detection of reactive antibodies to M. bovis by Indirect Enzyme-Linked Immunosorbent Assay (iELISA) identifies if there was an exposure to the microorganism. The current study aimed to optimize an iELISA from M. bovis total cell antigen, applying it to bovine serum samples, and to evaluate risk factors. Serum samples were obtained from 400 cows from 17 herds from Southeast Brazil. In the optimization of iELISA, the following was established: 2 μg/mL of antigen, sera dilution 1:300, and conjugate dilution 1:15000. The frequency was 62.3% (249/400) of reactive animals and 100% (17/17) of reactive herds. Risk factors were: herds with more than 100 animals (OR= 3.1; CI= 95%); Holstein breed (OR= 72.5; CI= 95%); cows (OR= 29.7; CI= 95%); intensive breeding system (OR= 3.3; CI= 95%); associated small ruminant production (OR= 4.4; CI= 95%); milk production above 500L (OR= 2.9; CI= 95%); no quarantine (OR= 1.5; CI= 95%); mechanical milking (OR= 5.5; CI= 95%) and cases of mastitis (OR= 5.5; CI= 95%). The proposed iELISA was able to detect antibodies reactive to M. bovis in bovine serum. Knowledge of these risk factors can assist in the implementation of prophylactic measures.

摘要
牛支原体（Mycoplasma bovis）是一种可引发牛多种病理病症的高度传染性病原。采用间接酶联免疫吸附试验（Indirect Enzyme-Linked Immunosorbent Assay，iELISA）检测牛支原体反应性抗体，可判断动物是否存在该病原的暴露史。本研究旨在以牛支原体全细胞抗原优化iELISA检测方法，将其应用于牛血清样本检测，并开展感染危险因素评估。
本研究采集了巴西东南部17个牛群的400头奶牛的血清样本。在iELISA优化过程中，确定了如下实验参数：抗原使用浓度为2 μg/mL，血清稀释比例为1:300，酶标结合物稀释比例为1:15000。本次检测的反应性动物占比为62.3%（249/400），反应性牛群占比为100%（17/17）。
本研究鉴定出的感染危险因素包括：牛群存栏量超过100头（优势比（Odds Ratio，OR）=3.1；95%置信区间（Confidence Interval，CI））；荷斯坦奶牛品种（OR=72.5；95%CI）；经产母牛（OR=29.7；95%CI）；集约化养殖模式（OR=3.3；95%CI）；伴生小反刍动物养殖（OR=4.4；95%CI）；单头奶牛产奶量超过500L（OR=2.9；95%CI）；未实施检疫隔离（OR=1.5；95%CI）；机械挤奶（OR=5.5；95%CI）以及存在乳腺炎病例（OR=5.5；95%CI）。
本研究优化的iELISA可在牛血清中有效检测到针对牛支原体的反应性抗体。明确上述危险因素可为牛支原体感染的防控措施制定提供科学参考依据。