Development of Real Time PCR to Study Experimental Mixed Infections of T. congolense Savannah and T. b. brucei in Glossina morsitans morsitans

Tsetse flies are able to acquire mixed infections naturally or experimentally either simultaneously or sequentially. Traditionally, natural infection rates in tsetse flies are estimated by microscopic examination of different parts of the fly after dissection, together with the isolation of the parasite in vivo. However, until the advent of molecular techniques it was difficult to speciate trypanosomes infections and to quantify trypanosome numbers within tsetse flies. Although more expensive, qPCR allows the quantification of DNA and is less time consuming due to real time visualization and validation of the results. The current study evaluated the application of qPCR to quantify the infection load of tsetse flies with T. b. brucei and T. congolense savannah and to study the possibility of competition between the two species. The results revealed that the two qPCR reactions are of acceptable efficiency (99.1% and 95.6%, respectively), sensitivity and specificity and can be used for quantification of infection load with trypanosomes in experimentally infected Glossina morsitans morsitans. The mixed infection of laboratory Glossina species and quantification of the infection suggests the possibility that a form of competition exists between the isolates of T. b. brucei and T. congolense savannah that we used when they co-exist in the fly midgut.

采采蝇（tsetse fly）可通过自然或实验方式，同时或序贯获得混合感染。传统上，采采蝇的自然感染率通过解剖后对其不同组织部位进行镜检，并结合体内寄生虫分离操作进行估算。然而，在分子技术问世之前，学界难以对采采蝇体内的锥虫感染进行虫种鉴定，也无法量化其体内的锥虫载量。尽管实时定量聚合酶链反应（quantitative PCR, qPCR）成本较高，但该技术可实现DNA定量，且由于可实时可视化结果并完成验证，耗时更短。本研究评估了qPCR定量采采蝇感染布氏锥虫布氏亚种（Trypanosoma brucei brucei, T. b. brucei）与刚果锥虫草原亚种（Trypanosoma congolense savannah, T. congolense savannah）的感染负荷的应用效果，并探究了这两个物种间存在竞争关系的可能性。结果显示，这两套qPCR反应体系分别具有99.1%和95.6%的可接受扩增效率，且具备良好的灵敏度与特异性，可用于定量实验感染的刺舌蝇指名亚种（Glossina morsitans morsitans）体内的锥虫感染负荷。实验室饲养舌蝇物种的混合感染模型及其感染负荷定量结果表明，本研究所用的布氏锥虫布氏亚种与刚果锥虫草原亚种分离株在蝇类中肠内共存时，可能存在某种形式的竞争关系。