International Graduate Course of Veterinary Science and Technology, Faculty of Veterinary Science, Chulalongkorn University, Bangkok 10330, Thailand.
International Graduate Course of Veterinary Science and Technology, Faculty of Veterinary Science, Chulalongkorn University, Bangkok 10330, Thailand; Department of Parasitology, Faculty of Veterinary Medicine, Universitas Gadjah Mada, Indonesia.
Acta Trop. 2021 Aug;220:105957. doi: 10.1016/j.actatropica.2021.105957. Epub 2021 May 9.
Intraerythrocytic parasites are traditionally identified by the microscopic examination of Giemsa-stained blood smears. However, this method does not always allow for the identification of individual species in goat's RBCs. Moreover, its unreliability in detecting low levels of parasitemia makes it unsuitable for epidemiological investigations and leaves goat farms vulnerable to potential outbreaks. In the present study, a novel multiplex PCR (mPCR) targeting the cytochrome c oxidase subunit I (COI) gene was developed to detect and subsequently differentiate Plasmodium caprae, Theileria luwenshuni, and Babesia spp. The specificity of each primer set was assessed both in silico and with a panel of DNA samples from the hosts themselves and other goat hemoparasites. Amplicons generated from each pair of primers were 664, 555, and 320-bp for P. caprae, Babesia spp., and T. luwenshuni, respectively. These products were further confirmed by sequencing. Our novel mPCR reactions successfully demonstrated the accurate and simultaneous amplification of the three parasites' DNA samples. The current mPCR method showed no cross-amplification with unintended targets. The detection limit of the mPCR in this study was 10 parasites' DNA copies per reaction. The current mPCR was able to detect the minimum parasitemia of approximately 0.001%, 0.000005%, 0.00001% for P. caprae, Babesia spp. and T. luwenshuni, respectively. The diagnostic specificity in the detection of P. caprae and T. luwenshuni ranged from 94.9 to 100 %. The mPCR was further applied to a collection of field blood samples from five provinces in Thailand to validate its reliability and applicability. The results demonstrated the successful detection of P. caprae, Babesia spp. and T. luwenshuni in goat samples with the same sensitivity levels as conventional PCR methods. This study also confirmed the presence of T. luwenshuni and Babesia spp. in Thai goats. The current mPCR method offers an alternative for the diagnosis of P. caprae, T. luwenshuni, and Babesia spp., either single or under co-infection conditions, and for large-scale surveillance.
传统上，通过吉姆萨染色血涂片的显微镜检查来鉴定红细胞内寄生虫。然而，这种方法并不总是能够鉴定山羊 RBC 中的各个物种。此外，其在检测低水平寄生虫血症方面的不可靠性使其不适合流行病学调查，使山羊养殖场容易受到潜在疫情的影响。在本研究中，开发了一种针对细胞色素 c 氧化酶亚基 I（COI）基因的新型多重 PCR（mPCR），用于检测和随后区分山羊疟原虫、吕氏泰勒虫和巴贝斯虫。通过计算机模拟和来自宿主自身和其他山羊血液寄生虫的 DNA 样本的面板评估了每个引物对的特异性。从每个引物对产生的扩增子分别为 664、555 和 320-bp，用于 P. caprae、Babesia spp.和 T. luwenshuni。这些产物通过测序进一步得到证实。我们的新型 mPCR 反应成功地证明了三种寄生虫 DNA 样本的准确和同时扩增。当前的 mPCR 方法与非预期的靶标没有交叉扩增。在本研究中，mPCR 的检测限为每个反应 10 个寄生虫 DNA 拷贝。当前的 mPCR 能够检测到 P. caprae、Babesia spp.和 T. luwenshuni 的最低寄生虫血症约为 0.001%、0.000005%和 0.00001%。在检测 P. caprae 和 T. luwenshuni 时，诊断特异性范围为 94.9%至 100%。mPCR 进一步应用于来自泰国五个省份的一组现场血液样本中，以验证其可靠性和适用性。结果表明，在与常规 PCR 方法相同的灵敏度水平下，成功地从山羊样本中检测到 P. caprae、Babesia spp.和 T. luwenshuni。本研究还证实了吕氏泰勒虫和巴贝斯虫存在于泰国山羊中。当前的 mPCR 方法为 P. caprae、T. luwenshuni 和 Babesia spp.的诊断提供了一种替代方法，无论是单一感染还是混合感染，并且可用于大规模监测。
