# Identification of a Tsal1(52-75) salivary synthetic peptide to monitor cattle exposure to tsetse flies

Martin Bienvenu Somda, Sylvie Cornelie, Zakaria Bengaly, Françoise Mathieu-Daude, Anne Poinsignon, Emilie Dama, Jérémy Bouyer, Issa Sidibe, Edith Demettre, Martial Séveno, et al.

► To cite this version:

Martin Bienvenu Somda, Sylvie Cornelie, Zakaria Bengaly, Françoise Mathieu-Daude, Anne Poinsignon, et al.. Identification of a Tsal1(52-75) salivary synthetic peptide to monitor cattle exposure to tsetse flies. Parasites & Vectors, 2016, 9, pp.12. 10.1186/s13071-016-1414-8. hal-01511984

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# Identification of a Tsal1<sub>52-75</sub> salivary synthetic peptide to monitor cattle exposure to tsetse flies

Martin Bienvenu Somda<sup>1,2*</sup>, Sylvie Cornelie<sup>3</sup>, Zakaria Bengaly<sup>1</sup>, Françoise Mathieu-Daudé<sup>3</sup>, Anne Poinsignon<sup>3</sup>, Emilie Dama<sup>1,2</sup>, Jeremy Bouyer<sup>4,8</sup>, Issa Sidibé<sup>1,5</sup>, Edith Demettre<sup>6</sup>, Martial Seveno<sup>6</sup>, Franck Remoué<sup>3</sup>, Antoine Sanon<sup>7</sup> and Bruno Bucheton<sup>8</sup>

**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 Tsal1<sub>52-75</sub> synthetic peptide were shown to be specific of tsetse exposure in both naturally and experimentally exposed hosts. Nevertheless, anti-Tsal1<sub>52-75</sub> IgG responses were absent in animals exposed to high tsetse biting rates.

**Conclusions:** These results suggest that Tsal1<sub>52-75</sub> 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:** Synthetic Peptide, Biomarker of Exposure, Cattle, Tsetse Flies, African Animal Trypanosomosis

<footnote>* Correspondence: somdabienvenu@yahoo.fr</footnote>

<footnote n="1"><sup>1</sup>Centre International de Recherche-Développement sur l'Elevage en zone Subhumide (CIRDES), 01 BP 454 Bobo-Dioulasso 01, Burkina Faso</footnote>

<footnote n="2"><sup>2</sup>Université Polytechnique de Bobo-Dioulasso, 01 BP 1 091 Bobo-Dioulasso 01, Burkina Faso</footnote>

<footnote>Full list of author information is available at the end of the article</footnote>
## Background

African trypanosomosis, a parasitic vector-borne disease that constitutes a major constraint to development in sub-Saharan Africa, exists under two forms: Human African Trypanosomosis (HAT) known also as sleeping sickness and African Animal Trypanosomosis (AAT) or *Nagana*. Tsetse flies (*Glossina* spp.) are the cyclical vectors of the trypanosome species causing these diseases. Some 46 million cattle distributed over 10 million km² in 38 sub-Saharan African countries [1, 2] are estimated to be at risk of contracting AAT and hamper significantly the socioeconomic development of these African regions [3, 4]. Among the 38 tsetse-infested countries, 34 are amongst the poorest in the world [5] and have included tsetse and trypanosomosis as a constraint in their poverty reduction strategy papers under the heavily indebted poor countries initiative [6].

The main strategies used to control or eradicate AAT remain (i) chemoprophylaxis and chemotherapy with trypanocidal drugs, (ii) promoting trypanotolerant cattle, and (iii) tsetse control or eradication programmes [1]. The Pan African Tsetse and Trypanosomiasis Eradication Campaign (PATTEC) initiative promoting integrated control of AAT and large tsetse eradication campaigns are underway in Uganda, Kenya and Ethiopia in East Africa and in Ghana, Burkina Faso and Mali in West Africa, with the aim of improving breeding and agriculture by creating new tsetse free areas [7].

Entomological evaluation tools represent an important component of any vector based control programme to appropriately target implementation areas and to evaluate their efficacy in time [8]. To date, the conventional method used within tsetse eradication campaigns is to estimate tsetse fly densities with traps deployed at fixed or temporary sites [9]. Important constraints are nevertheless associated with this method. The deployment and monitoring of traps, most of the time in very large areas (20 km/day/person in walking) with poor accessibility, is costly and demanding in terms of human resources and logistics. It is also known that traps have a poor efficiency (below 1 % of the flies present in a 1 km² around the trap are captured daily) and are becoming even less efficient at low tsetse densities [8, 10, 11]. Furthermore, traps are generally set up in sentinel fixed sites and thus only provide an indirect estimate of cattle exposure to tsetse bites especially in agro-pastoral areas where herds are very mobile. Alternative methods based on the evaluation of the antibody (Ab) responses directed against bloodsucking arthropod salivary antigens have been developed in the last few years. During the blood meal, hematophagous arthropods inject a mixture of anti-haemostatic, anti-inflammatory and immunomodulatory molecules into the skin of their hosts. These molecules play a crucial role in achieving an effective

blood meal [12], but also in the establishment or not of pathogens into the vertebrate host [13]. The antigenic properties of these molecules have also been used to develop a range of immunoassays to detect associated specific Abs and to assess host exposure to a range of arthropod vectors of human pathogens [14].

Recent studies have shown that the human IgG response against whole salivary extracts (WSE) of several *Glossina* (G.) species (*G. morsitans* (m.) *morsitans*, *G. fuscipes* (f.) *fuscipes* and *G. palpalis* (p.) *gambiensis*) was correlated with human exposure to tsetse flies in different HAT endemic areas [15–18]. Similar results were observed in outbred cattle from a tsetse infested area in South-West Burkina Faso that were shown to harbor higher IgG responses than cattle in the North where tsetse flies are absent. These results were further confirmed in cows experimentally exposed to the bite of a range of *Glossina* species and other bloodsucking arthropods [19]. However, the use of WSE in immunoassays is likely impaired by the existence of potential cross-reactions with Abs directed against common saliva antigens that are shared by different arthropod species [20]. Other important drawbacks are the difficulty in achieving mass production of WSE in a standardized manner and the storage of these antigens over long periods. WSE are thus poorly suited for large scale studies [21, 22]. Resorting to recombinant salivary proteins has enabled to overcome some of these limitations and a number of immunoassays based on specific recombinant salivary proteins have been developed to detect exposure to a range of arthropods [23–27]. An alternative strategy has been to identify specific linear epitopes by *in silico* approaches in order to design peptides to be used as the immunoassay antigens. Production of short synthetic peptides at high purity can easily be entrusted to a private company; shipment and storage are facilitated as they can be lyophilised. In the last few years, such approaches were successfully applied to develop salivary biomarkers of human exposure to *Aedes aegypti* [28] and *Anopheles* (An.). *gambiae* [21, 22]. Recently, the IgG response to a synthetic peptide designed from the Tsetse Saliva Growth Factor-1 (Tsgf1) sequence, was shown to be specific of human exposure to tsetse flies [29] and was successfully used to assess the evolution of human tsetse contacts during a vector control intervention in Guinea [30].

In the present study, our aim was to design peptides that could be used to assess exposure of cattle to tsetse flies. An immuno-proteomic approach, using sera from animals experimentally exposed to several arthropod species and WSE from *G. m. submorsitans*, was first used to identify the most specific immunogenic salivary proteins to detect *Glossina* exposure. Several epitope prediction and protein conformation software were then
used in combination with Blast analysis to design linear
peptides within the identified proteins. Synthetic peptides
were finally produced and evaluated in an indirect-ELISA
test with serum samples from (i) cattle from tsetse free or
tsetse infested areas and (ii) cows experimentally exposed
to low or high numbers of tsetse flies.

**Methods**

Serum samples

All bovine serum samples used in this study were col-
lected from cattle outbred in different environmental
settings from Burkina Faso or were obtained by experi-
mental exposure of cows to several arthropod species
and are described in detail elsewhere [19]. Samples from
outbred cattle included 17 samples collected in a tsetse
free area in Northern Burkina Faso and 43 samples col-
lected in a tsetse infested area in the South-West part of
the country. Concerning experimentally exposed sera,
we included six samples collected from six cows indi-
vidually exposed weekly to the bite of G. m. submorsi-
tans, G. p. gambiensis, Amblyomma (A.) variegatum, An.
gambiae, Tabanidae spp. or Stomoxys spp. Samples used
in the present study were those collected at the end of
the exposure experiments, after 12 weeks for An. gam-
biae and 23 weeks for all other species. In addition we
also used 96 serum samples collected weekly from two
groups of four animals that were exposed to 50 G. m.
submorsitans flies twice a week during 11 weeks (high
exposure group) or 10 flies weekly during the same
period (low exposure group).

Production of whole salivary extracts

WSE were obtained from 10–12 day-old *G. m. submorsitans* uninfected males and females from the IRD/CIRAD colony in Montpellier (France). The tsetse saliva was collected as described previously [17] by a salivation technique that does not require the dissection of salivary glands to avoid the presence of non-salivary antigens in WSE. Briefly, tsetse flies (4–6 flies) were enclosed in 50 ml Falcon tubes closed by a mosquito net and placed above a drop of salivation buffer on warm slides. Buffer drops were collected after 10 min of salivation and were stored at –80 °C before use. Prior to electrophoresis, WSE were desalted and concentrated using the 2-D Clean-Up Kit (GE Healthcare, Germany) according to the manufacturer instructions. Protein concentrations of WSE were assessed by the Bradford method.

**Identification of salivary proteins**

Two-dimensional electrophoresis (2DE) and gel staining

For the first dimension electrophoresis, isoelectric focusing (IEF) was carried out with 60 µg of *G. m. submorsitans* WSE on 11 cm pH 3–11 non linear immobiline™ dry-Strips (GE Healthcare, Germany). Strips were rehydrated

for 10–20 h at room temperature with protein sample
made up to 170 µl in IEF buffer (7 M urea, 2 M thiourea,
4 % CHAPS, 0.2 % tergitol, 0.8 % IPG buffer, 1 % octylβ-
glucoside and 2 % DeStreak reagent). The IEF conditions
were: temperature 20 °C; current 50 µA per strip; 60 V
(step'n'hold) for 1 h; 500 V (gradient) for 1 h; 1 000 V
(gradient) for 1 h; 6 000 V (gradient) for 2 h and then 6
000 V holds to 30 000 Vhs. After the IEF, the strips were
reduced for 10 min with 65 mM DTT buffer pH 6.8
(50 mM Tris HCl pH 6.8, 6 M urea, 30 % Glycerol, 2 %
SDS) and alkylated for 15 min with 81 mM of iodoaceta-
mide buffer pH 8.8 (50 mM Tris HCl pH 8.8, 6 M urea,
30 % Glycerol, 2 % SDS). Then equilibrated strips in
pH 6.8 buffer (50 mM Tris HCl pH 6.8, 6 M urea, 30 %
Glycerol, 2 % SDS) were applied to 10-20 % SDS-PAGE
Tris–HCl gels (Criterion, Biorad) for second dimension
and sealed with agarose (1 % agarose low melting, 0.2 %
SDS and 150 mM Tris pH 6.8). 2DE-gels were run at 30 V
for 20 min and then 200 V for 55 min.

After the second dimension, a preparative 2DE-gel was
fixed for 20 min in 50 % ethanol/5 % acetic acid solu-
tion, and then 10 min in 50 % ethanol solution, washed
three times in ultrapure water. Finally, this gel was
stained with a colloidal blue solution (Fermentas, Saint-
Remy les Chevreuse, France) overnight and washed four
times in ultrapure water.

Identification of G. m. submorsitans salivary proteins by mass spectrometry

Visible spots after colloidal blue staining were manually
excised under a laminar flow hood and enzymatic in-gel
digestion was performed automatically (Tecan freedom
evo® proteomics) according to the Shevchenko modified
protocol [31].

Briefly, protein spots were digested using 150 ng of
trypsin, peptide extraction was performed using 5 sonic-
ation cycles of 2 min each and peptides were concen-
trated 1 h at 50 °C in a heat block. Peptide samples were
automatically spotted (Tecan freedom evo® proteomics).
For this step, 0.5 µl of peptide sample and 0.5 µl of α-
cyano-4-hydroxy-trans-cinnamic acid (a saturated solu-
tion prepared in acetonitrile/trifluoroacetic acid, 50:
0.1 %, vortexed, sonicated 30 s and microcentrifuged
30 s with a 1/3 dilution of the supernatant used as the
matrix) were deposited on a 384-well MALDI anchor-
ship target using the dry-droplet procedure [32] and air
dried at room temperature. Peptide samples were then
desalted using a 10 mM phosphate buffer and dried
again at room temperature. MALDI-TOF MS analysis
was performed using UltraFlex MALDI TOF-TOF mass
spectrometer (Brucker Daltonics, Bremen, Germany) in
the reflecton mode with a 26 kV accelerating voltage and
a 50 ns delayed extraction. The AutoXecute™ module of
Flexcontrol™ v3.0 (Bruker Daltonics) (laser power ranged
from 40 to 50 %, 600 shots) was used to acquire mass spectra. Spectra were analysed using FlexAnalysis™ software v3.0 (Bruker Daltonics) and calibrated internally with the autoproteolysis peptides of trypsin (m/z: 842.51; 1045.556; 2211.10). Peptides were selected in the mass range of 900–3000 Da.

Peptide Mass Fingerprint identification of proteins was performed by searching against the *Glossina* entries of either the Swiss-Prot or TrEMBL databases (http://www.expasy.ch) and by using the MASCOT v2.3 algorithm (http://www.matrixscience.com) with trypsin enzyme specificity and one trypsin missed cleavage allowed [33]. Carbamidomethyl was set as fixed cystein modification and oxidation was set as variable methionine modification for searches. A mass tolerance of 50 ppm was allowed for identification. Matching peptides with one missed cleavage were considered as pertinent when there were two consecutive basic residues or when arginine and lysine residues were in an acidic context. MASCOT scores higher than 47 were considered as significant (p < 0.05) for Swiss-Prot and TrEMBL (v 2011_04) database interrogations.

## Immunoblotting and specific proteins identification

The proteins separated by 2DE were then electro-transferred onto a PVDF (polyvinylidene difluoride, Biorad) membrane for western blotting as previously described [34]. The PVDF membrane was washed in Tris Buffer Saline (TBS) and then incubated in blocking buffer (TBS tween 0.05 % and 5 % dry milk) for 1 h 30 min. After washing three times with TBS tween 0.1 %, the membrane was incubated overnight at 4 °C with bovine serum diluted at 1/100 in TBS tween 0.05 % with 2.5 % dry milk. The membranes were then washed three times with TBS tween 0.1 % and three times with TBS, and then equilibrated with 2.5 % dry milk in TBS for 15 min. Mouse anti-bovine IgG conjugated to peroxidase (Sigma, St Louis, MO, USA) was added at a dilution of 1/15 000 in TBS tween 2.5 % with 2.5 % dry milk for 2 h 30 at room temperature. After addition of the secondary antibody, the membranes were washed four times with TBS tween 0.1 % and four times with TBS. The immunogenic proteins in membranes were revealed using the West Pico ECL (Pierce, Rockford, IL, USA) and exposed to XCLXposure films (Pierce) for 4 min.

Digital images of both western blotting and 2D gel were captured by scanning at 16 bits resolution under non saturating conditions, 300 dpi and stored in TIFF format. The proteins visualised on 2D gels were matched with immunogenic proteins detected by western blotting using SameSpots™ Software 3.3 (Nonlinear Dynamics) in order to identify immunogenic spots.

## Peptide design

The sequences of proteins of interest were downloaded on the ExPASy Proteomics Tools server (http://www.expasy.ch).

The signal peptides were predicted by SignalP 4.0 [35] and cleaved from the immature protein sequences. Secondary structures were then predicted with NetSurfP [36] and I-Tasser [37] servers. The surface exposure of proteins of interest has been viewed in 3D by the Pymol software (http://www.pymol.org).

The identification of putative linear B-cell epitopes of identified proteins was carried out with Bcepred [38], Bcpred [39] and Antigenicity plot server [40]. All epitopes that were identified by at least two out of three algorithms were selected. Peptides having a length less than 30 amino acids and including the maximum of epitopes were selected for further analyses. The specificity of peptide sequences to the *Glossina* genus was checked by requests on the NCBI Blast T non redundant databases (http://www.blast.ncbi.nlm.nih.gov/Blast.cgi). Synthetic peptides were synthesized and purified (95 %) by Genepep SA (St-Jean de Vedas, Montpellier, France). All peptides were shipped in lyophilised form and were then resuspended in ultrapure water (1 mg/ml) and stored as frozen aliquots.

## Evaluation of bovine IgG Ab level against synthetic peptides and WSE

The anti-peptide IgG responses were measured by indirect-ELISA, according to Poinsignon et al. [22] with minor modifications. Briefly, microtiter plates Maxisorp (Nunc, Roskilde, Denmark) were coated with peptide (20 µg/mL) in phosphate buffer saline (PBS) for 2 h 30 min at 37 °C. After three washes, plates were saturated with blocking buffer (Pierce, thermo scientific) 1 h at 37 °C. Sera diluted in PBS-tween 1 % (1/30 for Tsal152-75 and 1/10 for Tsal145-166) were incubated overnight at 4 °C. After five washes, sheep anti-bovine IgG conjugated to peroxidase (AbD Serotec, France) was added (in dilution 1/4000 for Tsal152-75 and 1/2000 for Tsal145-166) in PBS tween 1 % for 1 h 30 at room temperature. Colorimetric development was carried out using ABTS (2,2'-azino-bis (3-ethyl-benzthiazoline 6-sulfonic acid) diammonium) (Sigma St Louis, MO, USA) in 50 mM citrate buffer (pH 4) containing 0.003 % H₂O₂. Optical density (OD) was measured at 405 nm (45 min for Tsal152-75 and 1 h for Tsal145-166). IgG responses against WSE were also evaluated on the same samples as described previously [19]. Each test sample was analysed in duplicate in antigen wells and, in parallel, in a blank well containing no peptide solution or no WSE (ODn) to control non-specific reactions between the serum and the reagents. Individual results were expressed as ΔOD value calculated according to the formula ΔOD = ODx - ODn, where ODx represents the mean of individual OD in both antigen wells, as previously used [22].

## Statistical analysis

All data were analysed with GraphPad Prism5 software® (San Diego, CA, USA). After verifying that ΔOD values
did not assume Gaussian distribution using Shapiro-Wilk test, the non-parametric Mann-Whitney U-test was used for comparison of Ab levels between two independent groups. All differences were considered significant at *P* < 0.05.

## Results

### Identification of specific immunogenic salivary proteins of Glossina exposure

The sialome of *G. m. submorsitans* was investigated by 2D-gel electrophoresis separation of the WSE followed by colloidal blue staining (Fig. 1) and subsequent MALDI-TOF/MS identification. A total of 53 spots were observed with colloidal blue staining and could be picked for mass spectrometry analysis. Forty-seven spots were successfully identified by MALDI-TOF MS analysis providing a catalogue of seven salivary proteins (Table 1). For the spots of low molecular weight (< 25kD) no identification could be obtained because the amount of material was too scanty. In contrast, low molecular weight spots are much more intense in 2-D electrophoresis gels obtained with *G. m. morsitans* saliva extracts obtained by centrifugation of salivary glands [41]. These differences may be related to differences in the saliva composition of these two closely related subspecies but also to the saliva collection technique. Several spots with similar molecular weight but different isoelectric points led to the same identification suggesting the existence of isoforms for these proteins. All

spots were identified as related to *G. m. morsitans* salivary proteins [41]: the Tsetse salivary gland protein 1 (Tsal1); the Tsetse salivary gland protein 2 (Tsal2) and its two isoforms (Tsal2A and Tsal2B); the Tsetse Salivary Growth Factor 1 (TSGF-1); the Salivary Secreted Adenosine (SSA); the Adenosine Deaminase-related Growth Factor C (ADGF-C); the 5'-Nucleotidase family salivary protein (5'-nuc) and the Tsetse Antigen 5 (TAg5).

In order to identify *G. m. submorsitans* immunogenic salivary antigens that are specific of cattle exposure to tsetse flies, we established the 2D immunoblot profiles of serum samples from cows experimentally exposed to tsetse flies (*G. m. submorsitans*, *G. p. gambiensis*) or to other bloodsucking arthropods that are common in the study area (*A. variegatum*, *An. gambiae*, *Tabanidae* spp. or *Stomoxys* spp.). A number of spots were common between the two tsetse species but differences could be observed (Fig. 2). Whereas ADFG-C appears to be immunogenic in the cow exposed to *G. p. gambiensis*, this salivary protein did not react with the *G. m. submorsitans* serum. On the contrary, the *G. p. gambiensis* serum did not recognize any spot associated with the Tsal2 isoforms (Tsal2A and Tsal2B), TSGF-1, 5'-nuc and TAg5 families of salivary proteins. Importantly, cross reactions (although weak) were also observed with the sera of cows experimentally exposed to *Stomoxys* spp. or *An. gambiae* which recognised Tsal2, 5'-nuc, TAg5 and Tsal2, Tsal2A, 5'-nuc, TAg5 respectively (Table 2). Based

<figure><img src="image_3.png" /></figure>
<table><caption>**Table 1** *Glossina morsitans submorsitans* salivary secreted proteins identified by mass spectrometry. Database searches were performed against the *Glossina* entries of the SwissProt or TrEMBL databases with the MASCOT software. Molecular mass, pl and sequence coverage are shown. All the MASCOT scores are > 47 (p < 0.05)</caption><thead><tr><th rowspan="2">Abbreviation</th><th rowspan="2">Protein identification</th><th rowspan="2">Protein family</th><th rowspan="2">Swissprot-TrEMBL<br>accession number</th><th colspan="2">Mass (kDa)</th><th rowspan="2">pl</th><th rowspan="2">Coverage (%)</th><th rowspan="2">Mascot score</th><th rowspan="2">Length (amino acids)</th><th rowspan="2">Functions</th></tr><tr><th>Exp</th><th>Theo</th></tr></thead><tbody><tr><td>Tsal1</td><td>Tsetse salivary gland protein<br>1, G. m. m.</td><td>Endonuclease</td><td>D3TS87_GLOMM</td><td>46.155</td><td>45.613</td><td>5.06</td><td>13</td><td>79</td><td>399</td><td>Endonuclease activity and blood<br>meal digestion</td></tr><tr><td>Tsal2</td><td>Tsetse salivary gland protein<br>2, G. m. m.</td><td>Endonuclease</td><td>D3TMW5_GLOMM</td><td>45.550</td><td>43.956</td><td>5.74</td><td>28</td><td>68</td><td>388</td><td>Endonuclease activity and blood<br>meal digestion</td></tr><tr><td>Tsal2A</td><td>Tsetse salivary gland protein<br>2, isoform A, G. m. m.</td><td>Endonuclease</td><td>A3FMN3_GLOMM</td><td>44.601</td><td>44.002</td><td>6.16</td><td>22</td><td>76</td><td>388</td><td>Endonuclease activity and blood<br>meal digestion</td></tr><tr><td>Tsal2B</td><td>Tsetse salivary gland protein<br>2, isoform B, G. m. m.</td><td>Endonuclease</td><td>A3FMN4_GLOMM</td><td>44.567</td><td>43.968</td><td>5.74</td><td>20</td><td>68</td><td>388</td><td>Endonuclease activity and blood<br>meal digestion</td></tr><tr><td>TSGF-1</td><td>Tsetse Salivary Growth<br>Factor-1, G. m. m.</td><td>Salivary adenosine<br>deaminase (ADA)</td><td>D3TLK6_GLOMM</td><td>56.783</td><td>56.591</td><td>5.52</td><td>44</td><td>245</td><td>494</td><td>Vasolidation and platelet<br>anti-aggregating</td></tr><tr><td>SSA</td><td>Salivary Secreted Adenosine,<br>G. m. m.</td><td>Salivary adenosine<br>deaminase (ADA)</td><td>D3TQW6_GLOMM</td><td>41.309</td><td>41.222</td><td>9.94</td><td>12</td><td>82</td><td>349</td><td>Vasolidation and platelet<br>anti-aggregating</td></tr><tr><td>ADGF-C</td><td>Adenosine deaminase-related<br>growth factor C, G. m. m.</td><td>Salivary adenosine<br>deaminase (ADA)</td><td>DT3QW4_GLOMM</td><td>62.390</td><td>62.201</td><td>7</td><td>19</td><td>99</td><td>535</td><td>Vasolidation and platelet<br>anti-aggregating</td></tr><tr><td>5'-nuc</td><td>5'nucleotidase family salivary<br>protein, G. m. m.</td><td>5'nucleotidase/<br>Apyrase</td><td>D3TRV7_GLOMM</td><td>62.479</td><td>62.062</td><td>7.19</td><td>16</td><td>75</td><td>555</td><td>ATP-diphosphohydrolase activity</td></tr><tr><td>TAg5</td><td>Tsetse Antigen 5, G. m. m.</td><td>Antigen 5<br>(AG5) family</td><td>Q9NBA6_GLOMM</td><td>29.647</td><td>28.925</td><td>8.58</td><td>40</td><td>113</td><td>295</td><td>Hypersensitivity I reaction and<br>anti-hemostatic activity</td></tr></tbody></table>

G. m. m.: *Glossina morsitans morsitans*, Exp : experimental and Theo : Theoretical
<figure><img src="image_4.png" /></figure>

on these results we decided to focus the peptide design on Tsal1 and SSA sequences as these salivary antigens were recognised only by the sera of animals individually exposed to both tsetse species and did not react with the sera of animals bitten by the other arthropod species.

## Peptide design

Four and one sequences of 22 to 28 amino acid residues containing putative linear epitopes were identified from the *G. m. morsitans* Tsal1 and SSA sequences respectively (Additional file 1: Table S1). In order to avoid potential cross-reactivity with Abs against proteins from other bloodsucking arthropod species as well as from host

pathogens, these 5 peptides were submitted to the NCBI Blast T non redundant databases (Additional file 1: Table S1). The SSA<sub>65–92</sub> peptide showed some degree of homology with *Culex quinquefasciatus* and was therefore dropped out in further analyses. Similarly, among the 4 peptides identified to carry putative Tsal1 linear epitopes, we selected Tsal1<sub>52–65</sub> and Tsal1<sub>145–166</sub> as for the other two peptides, closest matches were obtained for organisms to which cattle may be naturally exposed (*Plasmodium yoelii yoelii* and *An. darlingi*). After checking for Ab accessibility of these two peptides at the surface of the Tsal1 protein using 3D models (Fig. 3), Tsal1<sub>52–75</sub> and Tsal1<sub>145–166</sub> were synthesised and tested against cattle serum samples.

<table><caption>**Table 2** *Glossina morsitans submorsitans* salivary secreted proteins recognised by cows exposed to tsetse and other hematophagous arthropods</caption><thead><tr><th>Arthropod species used for experimental exposure</th><th>Tsal1</th><th>Tsal2</th><th>Tsal2A</th><th>Tsal2B</th><th>TSGF-1</th><th>SSA</th><th>ADGF-C</th><th>5'-nuc</th><th>TAg5</th></tr></thead><tbody><tr><td><i>G. m. submorsitans</i></td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>-</td><td>+</td><td>+</td></tr><tr><td><i>G. p. gambiensis</i></td><td>+</td><td>+</td><td>-</td><td>-</td><td>-</td><td>+</td><td>+</td><td>-</td><td>-</td></tr><tr><td><i>Stomoxys</i> spp.</td><td>-</td><td>+</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>+</td><td>+</td></tr><tr><td><i>An. gambiae</i></td><td>-</td><td>+</td><td>+</td><td>-</td><td>-</td><td>-</td><td>-</td><td>+</td><td>+</td></tr><tr><td><i>Tabanidae</i> spp.</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td><i>A. variegatum</i></td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr></tbody></table>

'+' indicates *Glossina morsitans submorsitans* salivary proteins recognised on 2D gels by western blot with sera from animals exposed experimentally to several arthropod species
<figure><img src="image_5.png" /><figcaption>**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<sub>52-75</sub> and green for Tsal1<sub>145-166</sub></figcaption></figure>

## Ability of synthetic peptides to detect exposure to tsetse flies

The ability of the two synthetic peptide candidates to detect exposure of cattle to tsetse flies was first assessed on serum samples from cattle bred in different climatic zones in Burkina Faso. Serum samples from the

cows experimentally exposed to the none-tsetse species were also included as negative controls (Fig. 4). Anti-Tsal1<sub>52-75</sub> IgG responses were significantly higher (*P* = 0.009) in cattle from tsetse infested area as compared to those from tsetse free area, and were the lowest in the control animals exposed to tabanids, stable flies, mosquitoes or ticks. They also provided a better discrimination between animals from tsetse infested or free areas as compared to the response directed against WSE (*P* = 0.116). In contrast no significant differences in the IgG response specific to Tsal1<sub>145-166</sub> (*P* = 0.574) were observed according to the exposure status, and this peptide was dropped out in further analyses. This may be due to the fact that as shown in Fig. 3 the Tsal1<sub>145-166</sub> sequence is predicted to be involved in the formation of Tsal1 secondary structures, which may have altered its immunogenic properties.

Finally the performance of the Tsal1<sub>52-75</sub> peptide as a biomarker of exposure was further explored with experimental sera obtained from cows exposed to low or high regimen of tsetse bites (Fig. 5). In the low exposure group (10 flies/once a week), an early increase of the specific IgG response was observed after only three weeks of exposure and was maintained over the exposure period (11 weeks). Unexpectedly, very low IgG Ab levels were observed in the high exposure group (50 flies/twice a week), throughout the exposure period

<figure><img src="image_6.png" /><figcaption>**Fig. 4** Cattle IgG responses against WSE and, Tsal1<sub>52-75</sub> and Tsal1<sub>145-166</sub> 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 ΔOD 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)</figcaption></figure>
<figure><img src="image_7.png" /><figcaption>**Fig. 5** Monitoring anti-Tsal1<sub>52-75</sub> and anti-tsetse saliva antibody responses in cows experimentally exposed to low and high levels of tsetse bites.</figcaption></figure>

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

despite the fact that these animals mounted high IgG re-
sponses to WSE.

**Discussion**

In this study, immuno-proteomics and bioinformatics
tools were combined to design specific peptides as po-
tential biomarker candidates for evaluating cattle expos-
ure to tsetse flies. Indirect-ELISA tests using the
identified peptides as antigens were then performed on
sera from cattle exposed naturally or experimentally to
tsetse bites. The Tsal1<sub>52-75</sub> peptide appears as a promis-
ing candidate as anti-Tsal1<sub>52-75</sub> IgG responses were de-
tected in both naturally exposed animals and in cows
submitted to low tsetse exposure levels that where close
to the tsetse challenge observed in the study area [42].

A number of tests have been developed to assess host
exposure to tsetse bites. Early studies focused on the de-
tection of host Abs raised against WSE from several tse-
tse species: G. p. gambiensis to assess human exposure
in West Africa [18]; G. m. submorsitans to assess cattle
exposure in West Africa [19]; G. f. fuscipes to assess hu-
man exposure in Central Africa [16, 17]; and G. m. mor-
sitans to assess human exposure in East Africa [15].
These studies showed that proteins from the Tsal family
are major constituents of tsetse saliva, and induce strong
Ab responses in tsetse exposed hosts. These proteins
were thus considered as interesting candidates to de-
velop biomarkers of tsetse exposure. Consecutively, it
was shown that a G. m. morsitans Tsal1 recombinant
proteins could be used instead of WSE in mice and pigs
experimentally exposed to tsetse flies [15, 26]. Because
the production of Tsal recombinant proteins in large
quantities is difficult, possibly due to the DNA binding/
endonuclease activity of Tsal proteins [43], these authors
also developed a nanobody-based competitive immuno-
assay to detect anti-Tsal Abs [44]. The advantage of this
method is that the same test can be applied to a wide

range of hosts; nevertheless the test still requires the use
of WSE, the production/storage of which can be a limi-
tation in the context of laboratories from developing
countries. In the present study, we evaluated the im-
munogenic properties of G. m. *submorsitans* salivary an-
tigens in cows exposed to two tsetse species and to
other bloodsucking arthropods. Whereas G. m. *submor-
sitans* Tsal proteins were shown to be highly immuno-
genic, only anti-Tsal1 IgG Abs were specific to tsetse fly
exposure as immune cross-reactions with Tsal2 proteins
were observed in animals exposed to stable flies or An.
gambiae. The results presented in the present work
point out Tsal1 as the best salivary antigen candidate to de-
velop a highly specific biomarker of cattle exposure. Never-
theless, a study carried out on humans led to different
results [18] as both Tsal proteins were recognised by sera
from unexposed individuals. Instead this work led to the
identification of a specific epitope within the TSGF-1 saliv-
ary protein [29], which has now been validated to monitor
human exposure to G. p. gambiensis during a vector con-
trol campaign in Guinea [30]. It is noteworthy that in the
present study, Ab response was detected against TSGF-1
only in the cow exposed to G. m. submorsitans. This illus-
trates that results obtained in a given animal model cannot
always be extrapolated to another. These differences are
likely due (i) to the sequence diversity of salivary proteins
between the different tsetse species; (ii) to host species
specificities in immune recognition; but also (iii) to the
range/level of biting insects or pathogens to which tsetse
hosts are submitted to and that can vary greatly between
mammals or eco-climatic contexts. Hence, available sero-
logical tools, especially those relying on the recognition of a
limited number of epitopes (such as it is the case for re-
combinant proteins and synthetic peptides), should be care-
fully evaluated prior to implementation as specificity and
sensitivity of a given test may vary greatly according to the
different contexts.
Tsal1<sub>52-75</sub>-based immunoassays, appear as promising tools to assess cattle exposure in West Africa where *G. morsitans* and *G. palpalis* subspecies represent the main tsetse species. Because this peptide was designed from the *G. m. morsitans* Tsal1 sequence, it might also be applied more widely as suggested by the results obtained experimentally on mice and pigs with the *G. m. morsitans* r-Tsal1 protein [26]. A surprising result was the fact that in our experimental conditions, no Ab response to Tsal1<sub>52-75</sub> was observed in the group of cows submitted to intensive tsetse fly bites despite the fact these cows exhibited strong Ab responses against WSE. This suggests that single epitopes behave differently in terms of immunogenicity according to the exposure conditions. Noteworthy, r-Tsal1 indirect-ELISA tests [26] or Tsal specific monoclonal nanobodies [44] were also less efficient than WSE to discriminate between mice or pigs exposed to different biting regimens. According to our results, the anti-Tsal1<sub>52-75</sub> Ab response represents a biomarker of low exposure levels but is likely less useful to measure the intensity of cattle exposure. The mechanisms underlying this intriguing result are not yet understood but could be related to antigen specific B cell exhaustion or anergy induced by high antigenic stimulation levels. This is however, an interesting feature for a biomarker candidate as it suggests that the development of a qualitative Tsal1<sub>52-75</sub> synthetic peptide-based immunochromatographic rapid test to detect low tsetse exposure levels is a reachable goal.

Declaring tsetse free areas or detecting the possible re-
emergence or reintroduction of tsetse flies after inter-
ventions is an important aspect of tsetse eradication
campaigns [45]. Using tsetse traps only is challenging
because this entomological method is not sensitive, even
less when tsetse densities are low [8]. Hence in such
context, it underestimates the true tsetse density or in-
correctly concludes to the absence of flies. Serological
tests able to detect low exposure levels could thus repre-
sent important alternative and complementary tools.
Such tests could be used in the field on cattle herds or
sentinel animals that are mobile baits naturally attractive
for tsetse flies. Such sentinel animals are already com-
monly used in the frame of tsetse vector control cam-
paigns to monitor trypanosome infections, an indirect
marker of tsetse exposure. In our experimental condi-
tions, the bite by less than 30 flies over a period of three
weeks was sufficient to induce anti-Tsal1<sub>52-75</sub> Ab re-
sponses. Further studies evaluating different biting regi-
mens (number of flies, biting frequencies) as well as re-
challenge experiments in previously exposed animals are
required to determine more precisely the sensitivity of
Tsal1<sub>52-75</sub>-based immunoassays. Indeed anti-saliva Abs
were shown to be boosted by very low numbers of tsetse
bites in re-challenged mice and pigs [26]. It will also be

useful to determine the persistency of Tsal1<sub>52-75</sub> Ab
after an exposure to tsetse bites.

Conclusions

In conclusion, we identified a Tsal1 peptide whose Ab
response is specific of cattle tsetse fly exposure. The IgG
response directed to the Tsal1<sub>52-75</sub> synthetic peptide
could be a biomarker of low cattle exposure. These
are promising results in the framework of developing
simple Tsal1<sub>52-75</sub> based immunoassays (such as rapid
tests) to monitor the tsetse flies presence at low fly
densities or to detect early reemergence in previously
cleared areas.

**Additional file**

**Additional file 1: Table S1.** Synthetic peptides and putative linear epitopes from Glossina morsitans morsitans Tsal1 and SSA salivary proteins. (DOCX 31 kb)

Competing interests

The authors declare that they have no competing interests.

Authors’ contributions

MBS: immunological analysis, peptides design, statistical analysis, data interpretation and drafting of the manuscript. 5C: immunological analyses, peptides design, data interpretation and drafting of the manuscript. ZB: conception of the research idea, study design and its coordination. FMD: immunoblot assays and drafting of the manuscript. AP and FR: study design, implementation of immunological assays and reviewing the manuscript. ED: ELISA assays, statistical analysis and data interpretation. IS, JB and AS: reviewing the manuscript. EDV and MS: mass spectrometry analysis and interpretation. BB: designing of the study, co-coordination of the study and drafting of the manuscript. All authors read and approved the final version of the manuscript.

Acknowledgements

This work was supported by the French Ministry of Foreign Affairs (AIRES-Sud project), the "Institut de Recherche pour le Développement" (IRD), the International Atomic Energy Agency (IAEA) and the International Foundation for Science (IFS). Martin Bienvenu Somda received a PhD grant from the "Service de Coopération et d'Action Culturelle" from the French Embassy in Burkina Faso.

Author details

¹Centre International de Recherche-Développement sur l'Elevage en zone Subhumide (CIRDES), 01 BP 454 Bobo-Dioulasso 01, Burkina Faso. ²Université Polytechnique de Bobo-Dioulasso, 01 BP 1 091 Bobo-Dioulasso 01, Burkina Faso. ³Institut de Recherche pour le Développement (IRD), Unité Mixte de Recherche 224, Maladies Infectieuses et Vecteurs: Ecologie, Génétique, Evolution et Contrôle (MIVEGEC), Montpellier 34394 Cedex 5, France. ⁴CIRAD, UMR CIRAD-INRA Contrôle des Maladies Animales, Campus International de Baillarguet, F34398 Montpellier, France. ⁵Pan African Tsetse and Trypanosomosis Eradication Campaign (PATTEC), Projet de Création de Zones Libérées Durablement de Tsé-tsé et de Trypanosomoses (PCZLD), Bobo-Dioulasso, Burkina Faso. ⁶Institut de Génomique Fonctionnelle, CNRS UMR 5203, INSERM U1191, UM1, UM2, Plate-forme de Protéomique Fonctionnelle CNRS UMS BioCampus 3426, 34094 Montpellier, France.

⁷Université de Ouagadougou, UFR/SVT, Laboratoire d'Entomologie Fondamentale et Appliquée (LEFA), BP 9499 Ouagadougou 06, Burkina Faso.

⁸Institut de Recherche pour le Développement, Unité Mixte de Recherche IRD-CIRAD 177, Interactions hôtes-vecteurs-parasites dans les maladies dues aux Trypanosomatidae, Campus International de Baillarguet, Montpellier 34398 Cedex 5, France.
Received: 18 November 2015 Accepted: 1 March 2016

Published online: 15 March 2016

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