National Institute of Animal Biotechnology, Hyderabad, India.
Department of Parasitology, College of Veterinary Science, Rajendra Nagar, Hyderabad, India.
Sci Rep. 2018 Oct 18;8(1):15441. doi: 10.1038/s41598-018-33721-3.
Theileria annulata is an intracellular parasite that causes active and latent forms of bovine theileriosis. Diagnosis of the disease is primarily based on traditional methods such as microscopy, however, PCR based methods have proven to be superior in the absence of clear disease symptoms. However, diagnosis is difficult in cases of lower parasitaemia by conventional PCR. Hence, a rapid and sensitive method which can detect early infection and low parasite load is required. Therefore, we have developed an absolute quantification based real-time PCR (qPCR) assay. Reference standard curve using recombinant plasmids of a host (hprt) and a parasite gene (tasp) was constructed, and the assay was initially standardised using in vitro T. annulata cell lines. Further, 414 blood samples from suspected theileriosis cases were also evaluated using qPCR. The assay can estimate host to parasite ratios, calculate parasitaemia and treatment effectiveness in the clinical cases of theileriosis. In comparison with the conventional PCR results, 44 additional positive cases were found. Therefore, the assay holds importance in a clinical setting due to its ability to quantify the parasite load in clinical samples. It may be further used in distinguishing active and latent theileriosis infections and detection of drug resistance in the field.
环形泰勒虫是一种细胞内寄生虫，可引起牛泰勒虫病的活动期和潜伏期。该疾病的诊断主要基于显微镜等传统方法，但在没有明确疾病症状的情况下，基于 PCR 的方法已被证明更优越。然而，在常规 PCR 检测到较低寄生虫血症时，诊断变得困难。因此，需要一种能够检测早期感染和低寄生虫载量的快速、敏感的方法。因此，我们开发了一种基于绝对定量的实时 PCR（qPCR）检测方法。使用宿主（hprt）和寄生虫基因（tasp）的重组质粒构建了参考标准曲线，并使用体外环形泰勒虫细胞系对该检测方法进行了初步标准化。此外，还使用 qPCR 评估了来自疑似泰勒虫病病例的 414 份血液样本。该检测方法可以估计宿主与寄生虫的比例，计算临床泰勒虫病病例中的寄生虫血症和治疗效果。与常规 PCR 结果相比，发现了 44 个额外的阳性病例。因此，由于该检测方法能够定量检测临床样本中的寄生虫载量，因此在临床环境中具有重要意义。它可以进一步用于区分活动期和潜伏期的泰勒虫感染以及在现场检测耐药性。
