Department of Veterinary Parasitology, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana, 141004, Punjab, India.
Department of Veterinary Parasitology, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana, 141004, Punjab, India.
Ticks Tick Borne Dis. 2022 May;13(3):101937. doi: 10.1016/j.ttbdis.2022.101937. Epub 2022 Mar 8.
Two multiplex SYBR Green based real-time PCR assays were standardized and evaluated to detect DNA from four canine haemoparasites (Babesia gibsoni, Babesia vogeli, Ehrlichia canis and Hepatozoon canis), along with internal controls from dogs from selected districts of Punjab state, India. Amplicons of 126 bp, 337 bp, 234 bp and 106 bp corresponding to B. gibsoni (18S rRNA gene), B. vogeli (18S rRNA gene), E. canis (virB9 gene), and H. canis (18S rRNA gene) were obtained, without any non-specific amplification. Microscopic evaluation of 200 blood samples from dogs revealed the prevalence of B. gibsoni, E. canis and H. canis as 1.5%, 1.5% and 1.0%, respectively, while with the multiplex real-time PCR assays the values for B. gibsoni, B. vogeli, E. canis, and H. canis were 8.0%, 1.5%, 3.5% and 23.5%, respectively, with concurrent infections of B. gibsoni and H. canis (3.5%); E. canis and H. canis (2.0%) and B. gibsoni, B. vogeli, E. canis, and H. canis (0.5%). The diagnostic sensitivity of the multiplex real-time PCR assays with respect to microscopy in the detection of B. gibsoni, E. canis and H. canis was 100% while the specificity for B. gibsoni, B. vogeli, E. canis, and H. canis was 93%, 100%, 98% and 77%, respectively, revealing the respective strength of agreement as ″fair″, ″slight″, ″moderate″ and ″slight″ by kappa value statistics, and the data were statistically significant, for detection of B. gibsoni and E. canis infections, by Fisher's exact test. The analytical sensitivity of the multiplex PCR assays in detection of DNAs was 8.59 × 10 and 9.9 × 10 copies for B. vogeli and E. canis, respectively, and 1.15 × 10 and 3.41 × 10 copies for B. gibsoni and H. canis, respectively. Assessment of risk factors viz. age, sex, breed, season and locations showed no significant association with the prevalence of these haemoparasites except for B. vogeli, E. canis and H. canis where significant associations were found for location, age and breed, respectively by multiplex real-time PCR assays.
两种基于多重 SYBR Green 的实时 PCR 检测方法被标准化并评估，用于检测来自印度旁遮普邦选定地区的犬只中的四种犬血液寄生虫（吉氏巴贝斯虫、 vogeli 巴贝斯虫、犬埃立克体和犬肝孢子虫）的 DNA，以及犬的内参。获得了对应于 B. gibsoni（18S rRNA 基因）、B. vogeli（18S rRNA 基因）、E. canis（virB9 基因）和 H. canis（18S rRNA 基因）的 126 bp、337 bp、234 bp 和 106 bp 的扩增子，没有任何非特异性扩增。对 200 份犬血样的显微镜评估显示，B. gibsoni、E. canis 和 H. canis 的流行率分别为 1.5%、1.5%和 1.0%，而使用多重实时 PCR 检测方法，B. gibsoni、B. vogeli、E. canis 和 H. canis 的流行率分别为 8.0%、1.5%、3.5%和 23.5%，同时还存在 B. gibsoni 和 H. canis（3.5%）、E. canis 和 H. canis（2.0%）以及 B. gibsoni、B. vogeli、E. canis 和 H. canis（0.5%）的混合感染。多重实时 PCR 检测方法在检测 B. gibsoni、E. canis 和 H. canis 方面的诊断灵敏度为 100%，而对 B. gibsoni、B. vogeli、E. canis 和 H. canis 的特异性分别为 93%、100%、98%和 77%，kappa 值统计显示各自的一致性强度为“公平”、“轻微”、“中度”和“轻微”，通过 Fisher 精确检验，检测 B. gibsoni 和 E. canis 感染的数据具有统计学意义。多重 PCR 检测方法在检测 DNA 方面的分析灵敏度分别为 8.59×10 和 9.9×10 拷贝的 B. vogeli 和 E. canis，以及 1.15×10 和 3.41×10 拷贝的 B. gibsoni 和 H. canis。对年龄、性别、品种、季节和地点等风险因素的评估显示，除了 B. vogeli、E. canis 和 H. canis 之外，这些血液寄生虫的流行率与这些风险因素之间没有显著关联，而通过多重实时 PCR 检测方法，发现了 B. vogeli、E. canis 和 H. canis 与地点、年龄和品种之间存在显著关联。
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