Guimaraes Ana M S, Santos Andrea P, Timenetsky Jorge, Bower Leslie P, Strait Erin, Messick Joanne B
1Joanne B. Messick, Department of Comparative Pathobiology, Purdue University, 625 Harrison Street, VPRB, West Lafayette, IN 47909.
J Vet Diagn Invest. 2014 Mar;26(2):203-12. doi: 10.1177/1040638713520542. Epub 2014 Feb 20.
The aims of the current study were to identify Mycoplasma suis antigens and develop a multiplex microbead immunoassay (MIA). A M. suis-expression library was screened for immunogens using sera from infected pigs. Based on bioinformatics, putative antigens were identified within positive inserts; gene fragments were expressed and purified as polyhistidine fusion proteins, and immunoreactivity was confirmed by Western blot. Selected antigens were used to develop a MIA. Sera from noninfected and infected pigs were used to set the median fluorescent intensity (MFI) cutoffs and as positive controls, respectively. Assay specificity was tested using sera from pigs seropositive for other pathogens (2 different pigs seropositive for each pathogen). Samples from 51 field pigs and 2 pigs during the course of acute (pig 1) and chronic (pig 2) infections were tested using MIA, indirect hemagglutination assay (IHA), and quantitative polymerase chain reaction (qPCR). Sixteen reactive plaques (52 genes) were detected. A heat-shock protein (GrpE), a nicotinamide adenine dinucleotide-dependent glyceraldehyde 3-phosphate dehydrogenase (GAPN), and 4 proteins from paralogous gene families (PGFs) were identified as antigens by Western blot. While GrpE, GAPN, and 1 PGF protein were strong antigens, the others were not suitable as MIA targets. A MIA using GrpE, GAPN, and the strongly reactive PGF protein was developed. Cross-reactivity with sera from pigs infected with Mycoplasma hyopneumoniae, Porcine circovirus-2, Porcine parvovirus, Porcine reproductive and respiratory syndrome virus, and Porcine respiratory coronavirus with this MIA was not observed. Pig 2 was consistently positive by MIA and qPCR, whereas pig 1, initially negative, seroconverted before becoming qPCR positive. Only 2 samples (from pig 1) were IHA positive. Five (9.8%) field samples were qPCR positive and 40 (78.43%) were positive for all 3 MIA antigens; however, all were IHA negative. In summary, the MIA is specific and more sensitive than qPCR and IHA, providing simultaneous evaluation of antibody response to M. suis antigens.
本研究的目的是鉴定猪支原体抗原并开发一种多重微珠免疫测定法（MIA）。使用感染猪的血清对猪支原体表达文库进行免疫原筛选。基于生物信息学，在阳性插入片段中鉴定出推定抗原；将基因片段表达并纯化作为多聚组氨酸融合蛋白，并通过蛋白质印迹法确认免疫反应性。使用选定的抗原开发一种MIA。分别使用未感染和感染猪的血清来设定中位荧光强度（MFI）临界值并作为阳性对照。使用感染其他病原体的猪的血清（每种病原体2头不同的血清阳性猪）测试测定法的特异性。使用MIA、间接血凝试验（IHA）和定量聚合酶链反应（qPCR）对51头猪场猪以及2头处于急性感染期（猪1）和慢性感染期（猪2）的猪的样本进行检测。检测到16个反应性噬菌斑（52个基因）。通过蛋白质印迹法鉴定出一种热休克蛋白（GrpE）、一种烟酰胺腺嘌呤二核苷酸依赖性甘油醛3 - 磷酸脱氢酶（GAPN）以及来自旁系同源基因家族（PGF）的4种蛋白质作为抗原。虽然GrpE、GAPN和1种PGF蛋白是强抗原，但其他蛋白不适合作为MIA靶点。开发了一种使用GrpE、GAPN和强反应性PGF蛋白的MIA。未观察到该MIA与感染猪肺炎支原体、猪圆环病毒2型、猪细小病毒、猪繁殖与呼吸综合征病毒以及猪呼吸道冠状病毒的猪的血清发生交叉反应。猪2通过MIA和qPCR始终呈阳性，而猪1最初为阴性，在qPCR呈阳性之前发生了血清转化。只有2个样本（来自猪1）IHA呈阳性。5个（9.8%）猪场样本qPCR呈阳性，40个（78.43%）对所有3种MIA抗原呈阳性；然而，所有样本IHA均为阴性。总之，该MIA具有特异性，并且比qPCR和IHA更敏感，可同时评估对猪支原体抗原的抗体反应。
