Department of Veterinary Pathobiology, College of Veterinary Medicine, Oklahoma State University, 250 McElroy Hall, Stillwater, OK, 74078, USA.
Oklahoma Animal Disease Diagnostic Laboratory, College of Veterinary Medicine, Oklahoma State University, 1950 W Farm Rd, Stillwater, OK, 74078, USA.
Vet Parasitol. 2021 Apr;292:109413. doi: 10.1016/j.vetpar.2021.109413. Epub 2021 Mar 15.
Cytauxzoonosis is a tick-borne disease of domestic cats with high mortality and narrow therapeutic window, particularly in the southcentral and southeastern United States. The causative agent is the apicomplexan protozoal parasite Cytauxzoon felis and is primarily transmitted by Amblyomma americanum, the lone star tick. Currently there is no vaccine available to prevent cytauxzoonosis and treatment is often ineffective if not initiated early enough in the course of disease. Early diagnosis and therapeutic intervention are therefore crucial for the survival of infected cats. Several methods are available for diagnosis of cytauxzoonosis, with PCR being the most sensitive. However, current PCR assays, which employ double-stranded DNA intercalating dyes to detect C. felis infection, have inherent limitations such as the potential for false positive detection of non-specific amplification products and inability to provide absolute quantification of parasite load. The objective of this study was to develop a probe-based droplet digital PCR (ddPCR) assay capable of detection and quantification of C. felis load over time and during treatment. The C. felis ddPCR assay was able to (i) reliably detect and quantify C. felis DNA in clinical blood samples from cats with acute cytauxzoonosis and (ii) monitor clinical parasite load in response to anti-protozoal treatment through absolute quantification of C. felis DNA over time. When tested on blood samples from cats with experimental C. felis infection, the assay was able to detect infection in cats as early as 24 h prior to the development of clinical signs. In addition, we demonstrate that this probe-based design can be utilized in traditional real-time PCR systems, with similar detection capabilities as compared to ddPCR. The C. felis probe-based ddPCR was also able to detect infection in samples with lower parasite loads when compared to existing nested PCR assays, although these results were not significant due to small sample size. To the author's knowledge, this is the first reported probe-based ddPCR assay to detect Cytauxzoon felis infection in domestic cats.
细胞球虫病是一种由蜱传播的家猫疾病，死亡率高，治疗窗口狭窄，特别是在美国中南部和东南部。病原体是原生动物寄生虫细胞球虫猫属，主要由孤星蜱传播。目前尚无预防细胞球虫病的疫苗，且如果在疾病过程中不能及早开始治疗，治疗效果往往不佳。因此，早期诊断和治疗干预对于感染猫的存活至关重要。有几种方法可用于诊断细胞球虫病，PCR 是最敏感的方法。然而，目前的 PCR 检测方法，采用双链 DNA 嵌入染料来检测 C. felis 感染，存在固有局限性，如非特异性扩增产物的假阳性检测的可能性以及无法提供寄生虫负荷的绝对定量。本研究的目的是开发一种基于探针的数字 PCR（ddPCR）检测方法，能够随着时间的推移和治疗过程中检测和定量 C. felis 负荷。C. felis ddPCR 检测方法能够：（i）可靠地检测和定量急性细胞球虫病猫的临床血液样本中的 C. felis DNA；（ii）通过随着时间的推移对 C. felis DNA 进行绝对定量，监测抗原生动物治疗的临床寄生虫负荷。在对患有实验性 C. felis 感染的猫的血液样本进行测试时，该检测方法能够在临床症状出现之前 24 小时检测到感染。此外，我们证明，与 ddPCR 相比，这种基于探针的设计可用于传统的实时 PCR 系统，具有类似的检测能力。与现有的巢式 PCR 检测方法相比，C. felis 基于探针的 ddPCR 还能够检测到较低寄生虫负荷的样本中的感染，尽管由于样本量小，这些结果并不显著。据作者所知，这是第一个报道的用于检测家猫细胞球虫感染的基于探针的 ddPCR 检测方法。
