Somda Martin Bienvenu, Cornelie Sylvie, Bengaly Zakaria, Mathieu-Daudé Françoise, Poinsignon Anne, Dama Emilie, Bouyer Jeremy, Sidibé Issa, Demettre Edith, Seveno Martial, Remoué Franck, Sanon Antoine, Bucheton Bruno
Centre International de Recherche-Développement sur l'Elevage en zone Subhumide (CIRDES), 01 BP 454, Bobo-Dioulasso 01, Burkina Faso.
Université Polytechnique de Bobo-Dioulasso, 01 BP 1 091, Bobo-Dioulasso 01, Burkina Faso.
Parasit Vectors. 2016 Mar 15;9:149. doi: 10.1186/s13071-016-1414-8.
The saliva of tsetse flies contains a cocktail of bioactive molecules inducing specific antibody responses in hosts exposed to bites. We have previously shown that an indirect-ELISA test using whole salivary extracts from Glossina morsitans submorsitans was able to discriminate between (i) cattle from tsetse infested and tsetse free areas and (ii) animals experimentally exposed to low or high numbers of tsetse flies. In the present study, our aim was to identify specific salivary synthetic peptides that could be used to develop simple immunoassays to measure cattle exposure to tsetse flies.
In a first step, 2D-electrophoresis immunoblotting, using sera from animals exposed to a variety of bloodsucking arthropods, was performed to identify specific salivary proteins recognised in cattle exposed to tsetse bites. Linear epitope prediction software and Blast analysis were then used to design synthetic peptides within the identified salivary proteins. Finally, candidate peptides were tested by indirect-ELISA on serum samples from tsetse infested and tsetse free areas, and from exposure experiments.
The combined immunoblotting and bioinformatics analyses led to the identification of five peptides carrying putative linear epitopes within two salivary proteins: the tsetse salivary gland protein 1 (Tsal1) and the Salivary Secreted Adenosine (SSA). Of these, two were synthesised and tested further based on the absence of sequence homology with other arthropods or pathogen species. IgG responses to the Tsal152-75 synthetic peptide were shown to be specific of tsetse exposure in both naturally and experimentally exposed hosts. Nevertheless, anti-Tsal152-75 IgG responses were absent in animals exposed to high tsetse biting rates.
These results suggest that Tsal152-75 specific antibodies represent a biomarker of low cattle exposure to tsetse fly. These results are discussed in the light of the other available tsetse saliva based-immunoassays and in the perspective of developing a simple serological tool for tsetse eradication campaigns to assess the tsetse free status or to detect tsetse reemergence in previously cleared areas.
采采蝇的唾液中含有多种生物活性分子的混合物，会在被叮咬的宿主中引发特定的抗体反应。我们之前已经表明，使用采采蝇指名亚种的全唾液提取物进行间接酶联免疫吸附测定（ELISA），能够区分：（i）来自采采蝇出没地区和无采采蝇地区的牛；（ii）实验中暴露于少量或大量采采蝇的动物。在本研究中，我们的目的是鉴定特定的唾液合成肽，这些肽可用于开发简单的免疫测定方法，以检测牛是否接触过采采蝇。
第一步，使用暴露于多种吸血节肢动物的动物血清进行二维电泳免疫印迹，以鉴定在被采采蝇叮咬的牛中识别出的特定唾液蛋白。然后使用线性表位预测软件和Blast分析，在已鉴定的唾液蛋白中设计合成肽。最后，通过间接ELISA对来自采采蝇出没地区和无采采蝇地区以及暴露实验的血清样本进行候选肽测试。
免疫印迹和生物信息学分析相结合，在两种唾液蛋白中鉴定出了五个带有推定线性表位的肽：采采蝇唾液腺蛋白1（Tsal1）和唾液分泌腺苷（SSA）。其中，基于与其他节肢动物或病原体物种不存在序列同源性，合成了两个肽并进行了进一步测试。在自然暴露和实验暴露的宿主中，对Tsal152 - 75合成肽的IgG反应均显示为采采蝇暴露所特有的。然而，在采采蝇叮咬率高的动物中不存在抗Tsal152 - 75 IgG反应。
这些结果表明，Tsal152 - 75特异性抗体代表牛低水平接触采采蝇的生物标志物。根据其他可用的基于采采蝇唾液的免疫测定方法，以及开发一种简单的血清学工具用于采采蝇根除运动以评估无采采蝇状态或检测先前清除区域中采采蝇重新出现的前景，对这些结果进行了讨论。
