Cuttell Leigh, Corley Sean W, Gray Christian P, Vanderlinde Paul B, Jackson Louise A, Traub Rebecca J
School of Veterinary Science, University of Queensland, Gatton QLD 4343 Australia.
Vet Parasitol. 2012 Sep 10;188(3-4):285-93. doi: 10.1016/j.vetpar.2012.03.054. Epub 2012 Apr 4.
Trichinella nematodes are the causative agent of trichinellosis, a meat-borne zoonosis acquired by consuming undercooked, infected meat. Although most human infections are sourced from the domestic environment, the majority of Trichinella parasites circulate in the natural environment in carnivorous and scavenging wildlife. Surveillance using reliable and accurate diagnostic tools to detect Trichinella parasites in wildlife hosts is necessary to evaluate the prevalence and risk of transmission from wildlife to humans. Real-time PCR assays have previously been developed for the detection of European Trichinella species in commercial pork and wild fox muscle samples. We have expanded on the use of real-time PCR in Trichinella detection by developing an improved extraction method and SYBR green assay that detects all known Trichinella species in muscle samples from a greater variety of wildlife. We simulated low-level Trichinella infections in wild pig, fox, saltwater crocodile, wild cat and a native Australian marsupial using Trichinella pseudospiralis or Trichinella papuae ethanol-fixed larvae. Trichinella-specific primers targeted a conserved region of the small subunit of the ribosomal RNA and were tested for specificity against host and other parasite genomic DNAs. The analytical sensitivity of the assay was at least 100 fg using pure genomic T. pseudospiralis DNA serially diluted in water. The diagnostic sensitivity of the assay was evaluated by spiking 10 g of each host muscle with T. pseudospiralis or T. papuae larvae at representative infections of 1.0, 0.5 and 0.1 larvae per gram, and shown to detect larvae at the lowest infection rate. A field sample evaluation on naturally infected muscle samples of wild pigs and Tasmanian devils showed complete agreement with the EU reference artificial digestion method (k-value=1.00). Positive amplification of mouse tissue experimentally infected with T. spiralis indicated the assay could also be used on encapsulated species in situ. This real-time PCR assay offers an alternative highly specific and sensitive diagnostic method for use in Trichinella wildlife surveillance and could be adapted to wildlife hosts of any region.
旋毛虫线虫是旋毛虫病的病原体，旋毛虫病是一种食源性人畜共患病，通过食用未煮熟的受感染肉类而感染。虽然大多数人类感染源自家庭环境，但大多数旋毛虫寄生虫在食肉和食腐野生动物的自然环境中传播。使用可靠且准确的诊断工具监测野生动物宿主中的旋毛虫寄生虫，对于评估野生动物向人类传播的患病率和风险至关重要。实时聚合酶链反应（PCR）检测方法此前已被开发用于检测商业猪肉和野生狐狸肌肉样本中的欧洲旋毛虫种类。我们通过开发一种改进的提取方法和SYBR绿检测法，扩大了实时PCR在旋毛虫检测中的应用，该检测法可检测来自更多种野生动物肌肉样本中的所有已知旋毛虫种类。我们使用伪旋毛虫或巴布亚旋毛虫乙醇固定幼虫，模拟野猪、狐狸、咸水鳄、野猫和澳大利亚本土有袋动物的低水平旋毛虫感染。旋毛虫特异性引物靶向核糖体RNA小亚基的保守区域，并针对宿主和其他寄生虫基因组DNA进行特异性测试。使用在水中连续稀释的纯伪旋毛虫基因组DNA，该检测方法的分析灵敏度至少为100飞克。通过在每10克每种宿主肌肉中分别接种伪旋毛虫或巴布亚旋毛虫幼虫，在每克1.0、0.5和0.1幼虫的代表性感染水平下评估该检测方法的诊断灵敏度，结果表明其能够检测到最低感染率的幼虫。对野猪和袋獾自然感染肌肉样本的现场样本评估显示，与欧盟参考人工消化方法完全一致（k值 = 1.00）。对实验感染旋毛虫的小鼠组织进行的阳性扩增表明，该检测方法也可用于原位检测包囊化种类。这种实时PCR检测方法为旋毛虫野生动物监测提供了一种高度特异性和灵敏的替代诊断方法，并且可适用于任何地区的野生动物宿主。
