Witold Stefański Institute of Parasitology, Polish Academy of Sciences, Twarda 51/55, 00-818, Warsaw, Poland.
Parasit Vectors. 2018 Nov 12;11(1):584. doi: 10.1186/s13071-018-3177-x.
Trichinella britovi is the second most common species of Trichinella that may affect human health. As an early diagnosis of trichinellosis is crucial for effective treatment, it is important to identify sensitive, specific and common antigens of adult T. britovi worms and muscle larvae. The present study was undertaken to uncover the stage-specific and common proteins of T. britovi that may be used in specific diagnostics.
Somatic extracts obtained from two developmental stages, muscle larvae (ML) and adult worms (Ad), were separated using two-dimensional gel electrophoresis (2-DE) coupled with immunoblot analysis. The positively-visualized protein spots specific for each stage were identified through liquid chromatography-tandem mass spectrometry (LC-LC/MS).
A total of 272 spots were detected in the proteome of T. britovi adult worms (Ad) and 261 in the muscle larvae (ML). The somatic extracts from Ad and ML were specifically recognized by T. britovi-infected swine sera at 10 days post infection (dpi) and 60 dpi, with a total of 70 prominent protein spots. According to immunoblotting patterns and LC-MS/MS results, the immunogenic spots recognized by different pig T. britovi-infected sera were divided into three groups for the two developmental stages: adult stage-specific proteins, muscle larvae stage-specific proteins, and proteins common to both stages. Forty-five Ad proteins (29 Ad-specific and 16 common) and thirteen ML proteins (nine ML-specific and four common) cross-reacted with sera at 10 dpi. Many of the proteins identified in Ad (myosin-4, myosin light chain kinase, paramyosin, intermediate filament protein B, actin-depolymerizing factor 1 and calreticulin) are involved in structural and motor activity. Among the most abundant proteins identified in ML were 14-3-3 protein zeta, actin-5C, ATP synthase subunit d, deoxyribonuclease-2-alpha, poly-cysteine and histide-tailed protein, enolase, V-type proton ATPase catalytic and serine protease 30. Heat-shock protein, intermediate filament protein ifa-1 and intermediate filament protein B were identified in both proteomes.
To our knowledge, this study represents the first immunoproteomic identification of the antigenic proteins of adult worms and muscle larvae of T. britovi. Our results provide a valuable basis for the development of diagnostic methods. The identification of common components for the two developmental stages of T. britovi may be useful in the preparation of parasitic antigens in recombinant forms for diagnostic use.
旋毛虫布氏亚种是第二常见的可能影响人类健康的旋毛虫种。由于早期诊断旋毛虫病对于有效治疗至关重要，因此识别成虫和肌肉幼虫的敏感、特异和共同抗原非常重要。本研究旨在揭示旋毛虫布氏亚种的阶段特异性和共同蛋白，这些蛋白可能用于特异性诊断。
使用二维凝胶电泳（2-DE）结合免疫印迹分析，从肌肉幼虫（ML）和成虫（Ad）两个发育阶段分离获得体提取物。通过液相色谱-串联质谱（LC-LC/MS）鉴定每个阶段特异性可视化的阳性蛋白点。
在旋毛虫成虫（Ad）的蛋白质组中检测到 272 个斑点，在肌肉幼虫（ML）中检测到 261 个斑点。在感染后 10 天（dpi）和 60 dpi，来自 Ad 和 ML 的体提取物被感染旋毛虫的猪血清特异性识别，共检测到 70 个明显的蛋白斑点。根据免疫印迹模式和 LC-MS/MS 结果，不同感染旋毛虫的猪血清识别的免疫原性斑点被分为两组，分别为成虫阶段特异性蛋白、肌肉幼虫阶段特异性蛋白和两个阶段共同的蛋白。45 个 Ad 蛋白（29 个 Ad 特异性和 16 个共同）和 13 个 ML 蛋白（9 个 ML 特异性和 4 个共同）在 10 dpi 时与血清发生交叉反应。在 Ad 中鉴定出的许多蛋白（肌球蛋白-4、肌球蛋白轻链激酶、副肌球蛋白、中间丝蛋白 B、肌动蛋白解聚因子 1 和钙网蛋白）参与结构和运动活性。在 ML 中鉴定出的最丰富的蛋白中，有 14-3-3 蛋白 zeta、肌动蛋白-5C、ATP 合酶亚基 d、脱氧核糖核酸酶-2-alpha、多半胱氨酸和组氨酸尾部蛋白、烯醇酶、V 型质子 ATP 酶催化亚基和丝氨酸蛋白酶 30。热休克蛋白、中间丝蛋白 ifa-1 和中间丝蛋白 B 在两个蛋白质组中均有鉴定。
据我们所知，本研究首次对旋毛虫布氏亚种成虫和肌肉幼虫的抗原蛋白进行了免疫蛋白质组学鉴定。我们的结果为开发诊断方法提供了有价值的基础。鉴定旋毛虫两个发育阶段的共同成分可能有助于制备用于诊断的重组寄生虫抗原。
