Identification of a Tsal152-75 salivary synthetic peptide to monitor cattle exposure to tsetse flies

Abstract

Background: 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.

Methods: 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.

Results: 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.

Conclusions: 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.

Keywords: African Animal Trypanosomosis; Biomarker of Exposure; Cattle; Synthetic Peptide; Tsetse Flies.

Fig. 1

2D gel profile (SDS-PAGE) of Glossina morsitans submorsitans secreted salivary proteins. Whole saliva extracts were run on 2DE gels and stained with colloidal blue. Fifty-three spots were analysed by mass spectrometry and 47 lead to an identification. Molecular weight markers (MW) are indicated on the left. Abbreviations: Tsal1 (Tsetse salivary gland protein 1), Tsal2 (Tsetse salivary gland protein 2), Tsal2A (Tsetse salivary gland protein 2, isoform A), Tsal2B (Tsetse salivary gland protein 2, isoform B), TSGF-1 (Tsetse Salivary Growth Factor 1), SSA (Salivary Secreted Adenosine), ADGF-C (Adenosine deaminase-related growth factor C), 5′-nuc (5′nucleotidase family salivary protein) and TAg5 (Tsetse Antigen 5)

Fig. 2

Glossina morsitans submorsitans immunogenic salivary proteins in cattle. G. m. submorsitans whole salivary extracts were run on 2D gels and transferred to PVDF membranes. Membranes were then incubated with sera from cows experimentally bitten by (a) G. m. submorsitans, (b) G. p. gambiensis, (c) An. gambiae and (d) Stomoxys spp. Molecular weight markers (MW) are indicated on the left

Fig. 3

Tsal1 3D prediction model. The image was generated by the Pymol software (http://www.pymol.org) from the most probable structures published on the I-Tasser server [37]. N-ter is the first amino acid of the protein and C-ter, the last. Candidate biomarker peptides are colored in red for Tsal1_52–75 and green for Tsal1_145–166

Fig. 4

Cattle IgG responses against WSE and, Tsal1_52–75 and Tsal1_145–166 peptides_. The IgG responses directed against whole saliva extracts (WSE) and the two candidate synthetic peptides were investigated in 43 animals from a tsetse infested area, 17 animals from a tsetse free area and four animals exposed experimentally to A. variegatum, An. gambiae, Tabanidae spp. or Stomoxys spp. (other arthropods). Individual ∆OD values are represented by empty circles. In the scatter plot, the horizontal bars indicate the median value for each group. Statistical significance between the different groups is indicated (non-parametric Mann–Whitney U-test)

Fig. 5

Monitoring anti-Tsal1_52–75 and anti-tsetse saliva antibody responses in cows experimentally exposed to low and high levels of tsetse bites. a Low exposure group (10 flies weekly) and (b) high exposure group (50 flies twice a week). Vertical bars above or below the curves are the standard errors of the group mean