Department of Epidemiology and Public Health Veterinary Institute, Federal Rural University of Rio de Janeiro, Rio de Janeiro, Brazil.
Department of Animal Parasitology, Veterinary Institute, Federal Rural University of Rio de Janeiro, Rio de Janeiro, Brazil.
Ticks Tick Borne Dis. 2018 Mar;9(3):556-562. doi: 10.1016/j.ttbdis.2018.01.013. Epub 2018 Feb 1.
A total of 300 blood samples of domiciliated dogs in rural and urban areas of southeast Rio de Janeiro State, Brazil, were used to compare the 18S ribosomal DNA region (18S rDNA) and the heat shock protein 70 kDa (hsp70) gene for molecular detection of Babesia vogeli and to perform a phylogenetic study comparing the two genes for B. vogeli classification. Using conventional polymerase chain reaction (cPCR) of 18S rDNA and hsp70 sequences, we were able to detect B. vogeli with the same sensitivity (96.15%) and specificity (99.63%). However, sequencing revealed one false positive (Rangelia sp.) for 18S rDNA that was not detected by hsp70. This is the first report of an organism closely related to the Rangelia vitalii parasite of dogs in Brazil. In the hsp70-cPCR and hsp70-qPCR comparison, 15.66% of samples were considered positive by quantitative (q)PCR, significantly more than was detected by cPCR (8.66%). In addition to the high conservation of the 18S rDNA, phylogenetic analysis showed that the hsp70 gene can be used to describe phylogenetic relationships between canine piroplasmids with more accuracy than 18S rDNA. According to these findings, the qPCR method has greater sensitivity than cPCR for detection of B. vogeli in naturally infected dogs. The hsp70-qPCR detection limit was 10 copies, with an efficiency of 100.30% and a determination coefficient (R) of 0.998. The development of this qPCR method provides a highly sensitive approach for B. vogeli molecular detection and a tool that is capable of quantifying parasitemia levels in whole blood samples from dogs. The primers and probes were designed to be specific for B. vogeli, though analytical specificity of the assay has not been tested in vitro with DNA of certain Babesia species that infect dogs. The hsp70 gene is a precise molecular marker for Babesia phylogeny, especially species that infect dogs.
本研究共采集了巴西东南部农村和城市地区 300 份犬血样，比较 18S 核糖体 DNA 区（18S rDNA）和热休克蛋白 70kDa（hsp70）基因，以分子检测犬巴贝斯虫（Babesia vogeli），并对两个基因进行系统发育研究，以对 B. vogeli 进行分类。使用常规聚合酶链反应（cPCR）对 18S rDNA 和 hsp70 序列进行检测，B. vogeli 的检测敏感性（96.15%）和特异性（99.63%）相同。然而，测序显示 18S rDNA 有 1 个假阳性（兰氏泰勒虫），而 hsp70 未检测到。这是巴西首次报道犬的 Rangelia vitalii 寄生虫密切相关的生物。在 hsp70-cPCR 和 hsp70-qPCR 比较中，15.66%的样本经定量（q）PCR 被认为是阳性，明显高于 cPCR（8.66%）。除了 18S rDNA 的高度保守性外，系统发育分析表明，hsp70 基因可用于更准确地描述犬巴贝斯虫之间的系统发育关系，比 18S rDNA 更准确。根据这些发现，qPCR 方法比 cPCR 更敏感，可检测自然感染犬中的 B. vogeli。hsp70-qPCR 的检测限为 10 拷贝，效率为 100.30%，决定系数（R）为 0.998。该 qPCR 方法的建立为犬巴贝斯虫的分子检测提供了一种高度敏感的方法，同时也是一种能够定量检测犬全血样本中寄生虫血症水平的工具。该引物和探针是专门针对 B. vogeli 设计的，尽管该方法的分析特异性尚未在体外用感染犬的某些巴贝斯虫属 DNA 进行测试。hsp70 基因是犬巴贝斯虫系统发育的精确分子标记，尤其是感染犬的种。
