囓齒類實驗動物內寄生蟲多重引子聚合酶鏈鎖反應之建立與傳統診斷方法之比較

Abstract

大小鼠的蟯蟲(Pinworm)、鼠三毛滴蟲(Tritrichomonas muris)以及鼠螺旋核原蟲(Spironucleus muris)是國內外各單位實驗動物須定期監測、預防與控制的內寄生蟲疾病。然而，目前傳統方法不但在內寄生蟲診斷上有效率與準確性的問題，且需犧牲動物，因此不符合動物福利原則。此研究針對實驗大小鼠的五種內寄生蟲，包含三種蟯蟲 (Syphacia obvelata 、 Syphacia muris 、 Aspiculuris tetraptera) 、鼠 三 毛 滴 蟲 (Tritrichomonas muris)及鼠螺旋核原蟲(Spironucleus muris)，以及大小鼠的管家基因(housekeeping gene)，建立了一個內寄生蟲多重引子聚合酶鏈鎖反應診斷法(Pinworm/Spironucleus/Tritrichomonas/Actin Multiplex PCR Assay)。此診斷法可特異性的增幅出目標基因，並能針對所有目標寄生蟲之偵測極限達到10 copies 的高敏感性。當模擬多種寄生蟲不等量共同感染的情況下，當兩種寄生蟲基因量相差高達100倍的情況下(104 vs. 102 copies)，或是三種寄生蟲量相差10倍的情況下(104 vs. 103 copies)，此診斷法仍能維持穩定的偵測效果。為比較此多重引子聚合酶鏈鎖反應與其他傳統診斷方法，收集來自11個單位共65個小鼠樣本與11大鼠樣本，以及來自5個單位共21個小鼠糞便樣本以及6個大鼠糞便樣本進行診斷。研究結果顯示，本多重引子聚合酶鏈鎖反應對於各寄生蟲皆具有高敏感性(97.2%-100%)與正確性(99%-100%)，優於其他的生前檢查法(敏感性：83.5%-100%；正確性：96.6%-100%)與死後檢查法(敏感性：75%-100%；正確性：92.1%-100%)。同時，此一試劑亦成功診斷出一件疑似蟯蟲感染的初期病例。本研究新開發的內寄生蟲多重引子聚合酶鏈鎖反應診斷法在不需要犧牲與干擾動物的前提下，可同時診斷五種內寄生蟲的感染情形，應為實驗動物疾病監控與管理上一個相當有用的工具。

Rodent pinworms, Spironucleus muris and Tritrichomonas muris are the endoparasites routinely monitored and excluded from laboratory animal colonies. Nevertheless, traditional diagnostic methods may not efficiently detect and accurately demonstrate the endoparasite infestation status even the animals are sacrificed and screened with postmortem diagnostic methods. In this study, a multiplex PCR assay was developed to target the rRNA genes to simultaneously detect and differentiate five endoparasites, including Syphacia obvelata, Syphacia muris, Aspiculuris tetraptera, Spironucleus muris, and Tritrichomonas muris, as well as a housekeeping gene (α-actin) in feces. Each primer set applied in this multiplex PCR is specific for each parasite target or rodent housekeeping gene. The multiplex PCR could diagnose an equivalent co-infection of pinworm, Spironucleus muris, and Tritrichomonas muris with a detection limit as low as 10 copies. Furthermore, dual infection with up to 100-fold differences in parasite loads (104 vs. 102 copies) and triple infection with 10-fold differences (104 vs. 103 copies) can also be detected. In comparison of traditional methods and the multiplex PCR assay, 65 mice and 11 rats from 11 colonies and another 21 mouse fecal samples and 6 rat fecal samples from 5 research facilities were screened for infestation of these endoparasites. The multiplex PCR exhibited the highest sensitivity and accuracy of 97.2%-100% and 99%-100%, respectively, compared to the traditional antemortem (sensitivity: 83.5%-100%; accuracy: 96.6%-100%) and postmortem methods (sensitivity: 75%-100%; accuracy: 92.1%-100%). In this study, a limited early outbreak of Syphacia obvelata in a SPF mouse colony was also diagnosed by this multiplex PCR assay. The Pinworm/Spironucleus/Tritrichomonas/Actin multiplex PCR assay developed in this study should be a powerful tool for diagnosing endoparasite infestations without sacrificing animals in routine health monitoring in the future.