The International Graduate Course of Veterinary Science and Technology (VST), Faculty of Veterinary Science, Chulalongkorn University, Bangkok, 10330, Thailand.
School of Agricultural Resources, Chulalongkorn University, Phayathai Rd., Pathumwan, Bangkok, 10330, Thailand.
Vet Parasitol. 2021 May;293:109431. doi: 10.1016/j.vetpar.2021.109431. Epub 2021 Apr 20.
Haemosporidian infections in domestic chickens (Gallus gallus domesticus) are not only widely prevalent but also cause economic loss. Diagnosis is usually made by microscopic examination; however, the method has several drawbacks such as requiring an experienced microscopist, being unreliable when parasitemia is low and being unable to accurately differentiate between co-infections from multiple parasite species. Therefore, the current extent of haemosporidian infections might be underestimated and neglected. We have developed a novel multiplex PCR assay to simultaneously detect and differentiate between four haemosporidian parasites: Leucocytozoon caulleryi, Leucocytozoon sabrazesi, Plasmodium juxtanucleare and Plasmodium gallinaceum. Primers in the present study specifically amplified the corresponding targets with no cross-species amplification detected. The multiplex PCR exhibited a significantly greater detection rate when compared with microscopic examination (p = 0.0001). The results demonstrate that the detection rate of multiplex PCR for L. sabrazesi, P. juxtanucleare, and P. gallinaceum are all greater than that of microscopic examination with p = 0.002, 0.0001 and 0.004, respectively. Co-infections were also detected more effectively by multiplex PCR. We applied the current method to field samples originating from Nan, Prachinburi, and Chachoengsao Provinces. The current study revealed that positive rates of haemosporidian parasites in chickens in the three study sites ranging from 39.5%-93.8%. The present assay offers a timesaving option for molecular diagnosis instead of using singleplex PCRs for detecting the parasites individually. Within a single reaction, this assay would be a useful tool for the detection of avian haemosporidian parasites either single or under co-infection conditions and for large-scale epidemiology studies.
家禽中血孢子虫的感染不仅非常普遍，还会造成经济损失。诊断通常通过显微镜检查来进行；然而，该方法存在几个缺点，例如需要有经验的显微镜专家，在寄生虫血症较低时不可靠，并且无法准确区分来自多种寄生虫物种的合并感染。因此，目前血孢子虫感染的程度可能被低估和忽视。我们开发了一种新的多重 PCR 检测方法，可同时检测和区分四种血孢子虫寄生虫：鸡白冠病、鸡泰勒虫、近核疟原虫和鸡疟原虫。本研究中的引物特异性扩增相应的靶标，没有检测到交叉物种扩增。与显微镜检查相比，多重 PCR 显示出显著更高的检测率（p=0.0001）。结果表明，多重 PCR 对鸡泰勒虫、近核疟原虫和鸡疟原虫的检测率均高于显微镜检查（p=0.002、0.0001 和 0.004）。多重 PCR 还能更有效地检测合并感染。我们将当前的方法应用于来自那空、巴真和差春骚府的现场样本。本研究显示，三个研究地点鸡的血孢子虫寄生虫阳性率为 39.5%-93.8%。与使用单个 PCR 分别检测寄生虫相比，当前的检测方法提供了一种节省时间的分子诊断选择。在单个反应中，该检测方法将成为检测单种或合并感染条件下禽血孢子虫寄生虫的有用工具，也适用于大规模的流行病学研究。
