0,0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 0,9 1/2 1/4 1/8 1/16 1/32 1/64 OD (4 05 n m ) TcWTL dilution T.congo and T.vivax infected bovine sera (immunoglobulin enriched) reactivity to TcWTL Negative sera Positive sera
TbPLA-1b antibody detection in immunoglobulin
enriched bovine sera (T.congolense/T.vivax infected)
TOUNKARA Magamba
1I
II
III
References :
1. Pillay et al, 2013; 2. Monic et al, in preparation; 3 Kralova et al, 2000Background
2.
African animal trypanosomiasis : Nagana
v
Major neglected animal disease caused by T. congolense, T.
vivax and T. b brucei.
Main hosts :
cow, sheep, goat.
v
Major livestock disease associated to
US $1-2 billon
losses/year, with more than 50 millions animals at risk (1).
v
No vaccine, inadequate diagnostics and therapeutics.
3.
Current diagnostic tools and tests
v
The disease control still relies on efficient diagnosis.
v
Current tests are facing a problem of sensitivity, they are
expensive and few targets available.
Need of new targets and new test development
1.
Trypanosomes
v
Unicellular and flagellate parasite from kinetoplastida order.
v
Deadly parasite, transmitted by blood-sucking insects :
Glossina sp and Tabanids and Stomoxes.
v
Develop and circulates in blood and lymph vessels with high
antigenic variation that makes difficult their detection.
a. Parasitological
b. Immunological
c. Molecular
Need special equipment, expensive, no specific
identification, very low sensitivity (except PCR).
Recombinant proteins for efficient African Animal Trypanosomiasis
(AAT) diagnosis : new candidates tests
TOUNKARA Magamba
1, Thonnus M
1, Bringaud F
1, Thévenon S
2, Berthier D
2, Boulangé A
2, Bengaly Z
3and Rivière L
1.
1
iMET team, UMR 5234-MFP, Université de Bordeaux, CNRS, Bordeaux, France ;
2Intertryp, UMR 17 CIRAD,
Montpellier, France ;
3CIRDES, Bobo-Dioulasso, Burkina Faso
Conclusion and Perspectives
Workflow
1
2
3
4
Candidates
Materials :
Trypanosomes infected mouse and bovine experimental
sera and bovine field sera are used in this work.
Fig 3 : Trypanosomes specific proteins
identified as potent diagnosis target.
Fig 2 : workflow for diagnostic test with each candidate.
Objectives :
Develop and standardize an easy, specific, sensitive and
inexpensive diagnostic test for animal trypanosomiasis.
Results
Recombinant expression
Bacterial Yeast + + pichiaC
andidate
1. Sequences analysis2. Immunoinformatics analysis
Bio-informatics
AAT diagnostic test development strategy using recombinant proteins
Candidates Single target Cocktail Chimera Diagnosis test
Three candidates are in
testing in the Lab. Two is
presented hereafter :
TbPLA-1b and TbGK.
Trypanosoma brucei phospholipase antibody detecting test
TbPLA-1b TbPLA are known as virulence factor. TbPLA-1b is currently study in the lab and is conserved in Trypanoma sp. 0,00 0,05 0,10 0,15 0,20 0,25 20 10 5 2,5 1,25 0,625 OD (4 05 n m ) TbPLA-1b concentration (µg/mL) TbPLA-1b antibody detecting test in T.congo and T.vivax infected bovine sera Negative sera Positive sera Negative sera Positive sera Milk BSA Fig 5 : Indirect ELISA test for antibody detection using different concentration of TbPLA-1b. Carbonate coating, blocking (Milk 5% and BSA 0.5%).
TbPLA-1b induces a specific antibodies during T. brucei infection in
mouse. Very low reactivity of T. congolense and T. vivax infected cattle sera to Tb PLA-1b. Specific antibody present in low concentration ?
Trypanosoma brucei glycerol kinase a key for AAT
diagnosis based on combination strategy
0,0 0,2 0,4 0,6 0,8 1,0 1,2 1,4 1,6 1,8 20 10 5 2,5 1,25 0,625 OD (4 05 n m ) TbPLA-1b concentration (µg/mL) TbPLA-1b antibody detection in T.congo and T.vivax infected bovine sera (immunoglobulin enriched) Negative sera Positive sera
Fig 6 : TbPLA-1b antibody detection by indirect ELISA in immunoglobulin
enriched bovine sera with NH2SO4. Left, TbPLA-1b in carbonate coating
and milk blocking ; right, positive control with trypanosomes total extract (WTL : Whole Trypanosome Lysate in PBS coating and BSA blocking).
High reactivity of positive sera to TbPLA-1b, proportional to its concentration,
equivalent to WTL reactivity. The result suggest a specific antibody presence but need to be confirmed. Immunoglobulin enrichment will be a limiting step for diagnosis.
Combination strategy : TbPLA-1b & TbGK (T.brucei Glycerol Kinase) ?
v glycerol-3-phosphate dependent ADP phosphorylation : ATP generation
v Very abundant and localized in the glycosome.
Fig 7 : T. brucei BSF
Glucose metabolism under anaerobic condition (3).
Fig 8 : TbGK antibody
detecting test. (Right up) Sequential epitopes
prediction using
BepiPred-2.0. (Right down) TbGK
antibody detection in T.bg,
T.congo and T.vivax infected
mouse sera. TbGK specific antibody as positive control.
Specific anti-GK antibody detected in virulent T.vivax infected mouse sera. Test confirmation with bovine sera (...). Combination TbGK_new candidate in the way.
T < 0.5 (high sensibility and low specificity) ; T > 0.5 (low sensibility and high specificity).
Conclusion:
v African animal trypanosomiasis diagnostic suffers from a lack of sufficiently
specific candidates for a field test. Here we tested two new candidates for AAT serological diagnostic. We demonstrated that specific anti-TbPLA-1b and TbGK antibodies are secreted during trypanosomes infection in mouse and bovine
(enriched sera, anti-TbPLA-1b).
To be continued… Confirm the specific antibody detection in trypanosomes infected
bovine sera.
v Because of large antigenic variation of trypanosomes, the alternative way for efficient AAT diagnostic will be a recombinant proteins combination.
Fig 4 : TbPLA-1b recognition by T.brucei
infected mouse sera by Western blot (2).
Perspectives
v New candidates screening
7 secreted
proteins 2 surface protein 3 cytosolic proteins
12 new candidates will be expressed and tested in single test. The most sensitives will be
mixed in cocktail to develop an efficient diagnostic test. For the test standardization, proteins
will be expressed in chimera.
PhD program
Fig 1 : ParaFrap PhD south program.MFP Bordeaux CIRDES Burkina Faso MFP Bordeaux MFP Bordeaux CIRDES Burkina Faso
6 Months 6 Months 6 Months
6 Months 1 year
