Zhang Qian, Dai Lu, Zhang Fuxian, Xu Zijie, Yang Xiangrui, Chu Hong, Yan Guangmou, Li Na, Li Fengyang, Lei Liancheng
College of Animal Science and Technology, Yangtze University, Jingzhou, China.
State Key Laboratory for Diagnosis and Treatment of Severe Zoonotic Infectious Diseases, Key Laboratory for Zoonosis Research of the Ministry of Education, Institute of Zoonosis, and College of Veterinary Medicine, Jilin University, Qinglong Road 818, Changchun, 130062, China.
Vet Res Commun. 2025 Jul 12;49(5):256. doi: 10.1007/s11259-025-10828-3.
Stenotrophomonas maltophilia (S. maltophilia) is a conditionally pathogenic bacterium around the world. In humans, it mainly harms patients with immunodeficient or chronic diseases, leading to high mortality rate of patients with pneumonia and bacteremia. In the veterinary medicine, it caused respiratory infections or mixed infections with other common pathogens in pigs. However, infection of S. maltophilia has been overlooked and the prevalence of S. maltophilia in porcine remains unknown due to a lack of diagnosis method.
In this study, an indirect ELISA was established using the purified Phospholipase C (PLC) protein of S. maltophilia SMFZ-01 strain as the coating antigen. Checkerboard titration was performed for the optimization of the coating concentration of the recombinant PLC (rPLC) protein, the dilution and incubation time of serum and the IgG-HRP secondary antibody. The specificity, repeatability and sensitivity of established ELISA was also optimized. The positive rate of S. maltophilia antibodies was calculated using 305 clinical porcine sera samples by either established indirect ELISA or a nested PCR.
rPLC was successfully purified and identified by Western blot. The optimal coating concentration of rPLC was 2.5 µg/mL. The optimal dilutions of serum samples and secondary antibody were 1:400 and 1:2500, respectively. The analytical sensitivity was 1:1600, with no cross-reaction with the positive sera of other common porcine pathogens. The intra-assay and inter-assay reproducibility coefficients of variation was less than 10%. Detection of 305 clinical porcine sera samples revealed a high positive rate by established ELISA (12.01%) and nested PCR (29.87%).
To our knowledge, this is the first study to develop an indirect ELISA for the detection and epidemiological analysis of S. maltophilia in porcine. Our findings indicate that S. maltophilia may be more prevalent in porcine populations than previously recognized, though further studies are needed to confirm this. The established indirect ELISA enhances our understanding of S. maltophilia infections in pig herds and provides valuable insights for designing future prevention and control strategies, along with considerations regarding the health and welfare of porcine.
嗜麦芽窄食单胞菌是一种在全球范围内具有条件致病性的细菌。在人类中，它主要危害免疫功能低下或患有慢性疾病的患者，导致肺炎和菌血症患者的死亡率很高。在兽医学中，它会引起猪的呼吸道感染或与其他常见病原体的混合感染。然而，由于缺乏诊断方法，嗜麦芽窄食单胞菌感染一直被忽视，其在猪群中的流行情况仍然未知。
在本研究中，以嗜麦芽窄食单胞菌SMFZ - 01菌株纯化的磷脂酶C（PLC）蛋白作为包被抗原来建立间接ELISA。通过棋盘滴定法优化重组PLC（rPLC）蛋白的包被浓度、血清的稀释度和孵育时间以及IgG - HRP二抗。还对所建立ELISA的特异性、重复性和敏感性进行了优化。使用305份临床猪血清样本，通过所建立的间接ELISA或巢式PCR计算嗜麦芽窄食单胞菌抗体的阳性率。
rPLC成功纯化并通过蛋白质免疫印迹法鉴定。rPLC的最佳包被浓度为2.5μg/mL。血清样本和二抗的最佳稀释度分别为1:400和1:2500。分析灵敏度为1:1600，与其他常见猪病原体的阳性血清无交叉反应。批内和批间重复性变异系数均小于10%。对305份临床猪血清样本的检测显示，所建立的ELISA阳性率较高（12.01%），巢式PCR阳性率为（29.87%）。
据我们所知，这是第一项开发用于检测和流行病学分析猪群中嗜麦芽窄食单胞菌的间接ELISA的研究。我们的研究结果表明，嗜麦芽窄食单胞菌在猪群中的流行可能比以前认识到的更为普遍，不过还需要进一步研究来证实这一点。所建立的间接ELISA增进了我们对猪群中嗜麦芽窄食单胞菌感染的了解，并为设计未来的预防和控制策略以及考虑猪的健康和福利提供了有价值的见解。
