Toxoplasma gondii is an obligate intracellular protozoan parasite that causes the most common parasitic zoonosis worldwide in multiples species of mammals and birds. Although free-range chickens may play a role as an important reservoir for T. gondii, there is no reliable and commercially available diagnostic test for this disease in chickens. In this study, we aimed to develop a novel Luciferase-linked Antibody Capture Assay (LACA) for the serodiagnosis of Toxoplasma infection in chickens. Recombinant nanoluciferase fused-T. gondii dense granule antigen 8 (rNluc-GRA8) was produced and applied to LACA assay as a diagnostic antigen. GRA8-LACA was tested with the sera from uninfected and experimentally infected chickens with T. gondii and other parasitic pathogens and showed unexpectedly high sensitivity (90.5%) and specificity (95.4%). Interestingly, E. coli lysate expressing rNluc-GRA8 could be applied in GRA8-LACA with 85.7% sensitivity and an increased specificity (96.9%) that gave better diagnostic performance compared to conventional ELISA. We applied our diagnostic system to examine 267 free-range chicken sera collected from 12 farms and 100 closed-house broiler chicken sera from local poultry abattoirs. The overall seroprevalence of toxoplasmosis in free-range chickens was 10.9% (95% CI: 10.6%–11.1%), while no positive case was found in broiler chickens. GRA8-LACA could be a useful diagnostic technique for T. gondii infection in chickens. The detection of T. gondii seropositive chickens in this study warns a potential risk of Toxoplasma transmission by the consumption of raw or undercooked chicken meat.

弓形虫是专性的细胞内原生动物寄生虫，在全世界多种哺乳动物和鸟类中引起最常见的寄生虫人畜共患病。尽管散养鸡可能是弓形虫的重要贮藏库，但尚无可靠且可商购的对该病进行诊断的方法。在这项研究中，我们旨在开发一种新颖的荧光素酶联结抗体捕获分析（LACA），用于鸡弓形虫感染的血清学诊断。产生了重组的纳米荧光素酶融合的弓形虫致密颗粒抗原8（rNluc-GRA8），并将其作为诊断抗原应用于LACA分析。用来自未感染和实验感染的弓形虫鸡的血清测试了GRA8-LACA和其他寄生性病原体，显示出乎意料的高灵敏度（90.5％）和特异性（95.4％）。有趣的是，表达rNluc-GRA8的大肠杆菌裂解物可用于GRA8-LACA中，灵敏度为85.7％，特异性更高（96.9％），与常规ELISA相比具有更好的诊断性能。我们应用诊断系统检查了从12个农场收集的267种散养鸡血清和从当地家禽屠宰场收集的100种封闭式肉鸡的血清。散养鸡的弓形虫病总体血清阳性率为10.9％（95％CI：10.6％-11.1％），而肉鸡未发现阳性病例。GRA8-LACA可能是鸡弓形虫感染的有用诊断技术。弓形虫的检测这项研究中的血清反应阳性的鸡通过食用生的或未煮熟的鸡肉警告了弓形虫传播的潜在风险。