Rickettsial Zoonoses Branch, Centers for Disease Control and Prevention, 1600 Clifton Rd., NE, MS H17-3, Atlanta, GA 30329, USA.
Rickettsial Zoonoses Branch, Centers for Disease Control and Prevention, 1600 Clifton Rd., NE, MS H17-3, Atlanta, GA 30329, USA; Department of Environmental Sciences, Emory University, Atlanta, GA 30329, USA.
Ticks Tick Borne Dis. 2020 Jul;11(4):101453. doi: 10.1016/j.ttbdis.2020.101453. Epub 2020 Apr 28.
The lone star tick (Amblyomma americanum) is the most common and abundant human-biting tick in the southeastern United States where spotted fever rickettsioses frequently occur. However, the role of this tick in transmitting and maintaining pathogenic and non-pathogenic spotted fever group rickettsiae (SFGR) remains poorly defined. This is partially due to the high prevalence and abundance of Rickettsia amblyommatis in most populations of A. americanum. Many molecular assays commonly employed to detect rickettsiae use PCR primers that target highly conserved regions in the SFGR so low abundance rickettsia may not be detected when R. amblyommatis is present. It is costly and inefficient to test for low abundance rickettsial agents with multiple individual specific assays even when they are multiplexed, as most samples will be negative. Real time PCR assays may also be hampered by inadequate limits of detection (LODs) for low abundance agents. We exploited the absence of an otherwise relatively SFGR-conserved genome region in R. amblyommatis to design a hemi-nested PCR-assay which has a sensitivity of 10 copies in detecting the presence of most SFGR, but not R. amblyommatis in DNA of infected lone star ticks. This deletion is conserved in 21 isolates of R. amblyommatis obtained from multiple states. We demonstrated the assay's utility by detecting a pathogenic SFGR, Rickettsia parkeri, in 15/50 (30 %) of field collected A. americanum ticks that were previously screened with conventional assays and found to be positive for R. amblyommatis. These co-infected ticks included 1 questing female, 6 questing nymphs, and 8 attached males. The high prevalence of R. parkeri among host-attached ticks may be due to several variables and does not necessarily reflect the risk of disease transmission from attached ticks to vertebrate hosts. This novel assay can provide accurate estimates of the prevalence of less common SFGR in A. americanum and thus improve our understanding of the role of this tick in the maintenance and transmission of the SFGR commonly responsible for human rickettsioses.
孤星蜱（Amblyomma americanum）是美国东南部最常见和丰富的人类叮咬蜱，那里经常发生斑点热立克次体病。然而，这种蜱在传播和维持致病性和非致病性斑点热群立克次体（SFGR）方面的作用仍未得到明确界定。这在一定程度上是由于大多数孤星蜱种群中莱姆病螺旋体的高流行率和丰富度。许多常用于检测立克次体的分子检测方法使用针对 SFGR 高度保守区域的 PCR 引物，因此当存在莱姆病螺旋体时，可能无法检测到低丰度的立克次体。即使将它们进行多重检测，使用多个个体特异性检测方法来检测低丰度的立克次体代理也是昂贵且低效的，因为大多数样本将呈阴性。实时 PCR 检测也可能受到低丰度试剂检测灵敏度不足（LOD）的限制。我们利用莱姆病螺旋体中相对 SFGR 保守基因组区域的缺失，设计了一种半巢式 PCR 检测方法，该方法在检测大多数 SFGR 的存在时具有 10 个拷贝的灵敏度，但在感染孤星蜱的 DNA 中不检测到莱姆病螺旋体。该缺失在从多个州获得的 21 株莱姆病螺旋体分离株中得到保守。我们通过检测先前用常规检测方法筛选为阳性的 50 只采集自野外的孤星蜱中 15/50（30%）的致病性 SFGR，帕克立克次体，证明了该检测方法的实用性。这些共感染的蜱包括 1 只游离雌蜱、6 只游离若虫和 8 只附着雄蜱。附着蜱中帕克立克次体的高流行率可能是由于多种变量引起的，并不一定反映从附着蜱向脊椎动物宿主传播疾病的风险。这种新的检测方法可以准确估计孤星蜱中较少见的 SFGR 的流行率，从而提高我们对立克次体在维持和传播常见致人类立克次体病的 SFGR 方面的作用的理解。
