Analyze Diet
Scientific reports2022; 12(1); 1308; doi: 10.1038/s41598-022-05356-y

Development of a microsphere-based immunoassay for the serological diagnosis of equine trypanosomosis.

Abstract: Trypanozoon infections in equids are caused by three parasite species in the Trypanozoon subgenus: Trypanosoma equiperdum, T. brucei and T. evansi. They are respectively responsible for infectious diseases dourine, nagana and surra. Due to the threat that Trypanozoon infection represents for international horse trading, accurate diagnostic tests are crucial. Current tests suffer from poor sensitivity and specificity, due in the first case to the transient presence of parasites in the blood and in the second, to antigenic cross-reactivity among Trypanozoon subspecies. This study was designed to develop a microsphere-based immunoassay for diagnosing equine trypanosomosis. We tested beads coated with eight Trypanosoma spp. recombinant antigens: enolase, GM6, PFR1, PFR2, ISG65, VSGat, RoTat1.2 and JN2118HU. Of these, GM6 was identified as the best candidate for the serological diagnosis of Trypanozoon infections in equids. Using a receiver operating characteristic (ROC) analysis on 349 equine sera, anti-GM6 antibodies were detected with an AUC value of 0.994 offering a sensitivity of 97.9% and a specificity of 96.0%. Our findings show that the GM6 antigen is a good target for diagnosing equine trypanosomosis using a microsphere-based immunoassay. This promising assay could be a useful alternative to the official diagnostic tool for equine trypanosomosis.
Publication Date: 2022-01-25 PubMed ID: 35079068PubMed Central: PMC8789838DOI: 10.1038/s41598-022-05356-yGoogle Scholar: Lookup
The Equine Research Bank provides access to a large database of publicly available scientific literature. Inclusion in the Research Bank does not imply endorsement of study methods or findings by Mad Barn.
  • Evaluation Study
  • Journal Article
  • Research Support
  • Non-U.S. Gov't

Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

The research article involves the development of a new diagnostic test for equine trypanosomosis using a microsphere-based immunoassay, with GM6 identified as the best-suited antigen. The new proposed diagnostic method reportedly has high sensitivity and specificity.

Background

  • Infections in equids (such as horses, donkeys and zebras) by trypanozoon parasites (Trypanosoma equiperdum, T. brucei and T. evansi) result in diseases such as dourine, nagana, and surra respectively. These diseases pose a significant threat to international horse trade.
  • The current diagnostic tests for equine trypanosomosis fall short due to their low sensitivity and specificity: sensitivity is affected by the transient presence of parasites in the blood, while specificity is compromised due to antigenic cross-reactivity among Trypanozoon sub-species.

Purpose of Research

  • The aim of this research was to develop a new diagnostic test for equine trypanosomosis using a microsphere-based immunoassay, in a bid to overcome the issues with the current diagnostic tests.

Methodology

  • The researchers tested beads coated with eight different recombinant antigens from the Trypanosoma species. The eight antigens tested were enolase, GM6, PFR1, PFR2, ISG65, VSGat, RoTat1.2, and JN2118HU.

Results

  • Among these eight antigens, GM6 emerged as the most suitable for the serological diagnosis of Trypanozoon infections in equids.
  • The effectiveness of GM6 was evaluated using a method called ‘receiver operating characteristic’ (ROC) analysis on samples from 349 equine sera.
  • The detection of anti-GM6 antibodies yielded an AUC (Area Under the ROC Curve) value of 0.994, which indicates an extremely high level of accuracy.
  • The sensitivity (ability to correctly identify those with the disease) of the GM6 antigen was calculated to be 97.9%, and its specificity (ability to correctly identify those without the disease) was calculated to be 96.0%. These high values indicate the diagnostic test’s effectiveness.

Conclusion

  • The research concluded that the GM6 antigen is a viable target for the diagnosis of equine trypanosomosis using a microsphere-based immunoassay.
  • This new immunoassay could present a valuable alternative to the currently employed diagnostic methods for equine trypanosomosis.

Cite This Article

APA
Verney M, Gautron M, Lemans C, Rincé A, Hans A, Hébert L. (2022). Development of a microsphere-based immunoassay for the serological diagnosis of equine trypanosomosis. Sci Rep, 12(1), 1308. https://doi.org/10.1038/s41598-022-05356-y

Publication

ISSN: 2045-2322
NlmUniqueID: 101563288
Country: England
Language: English
Volume: 12
Issue: 1
Pages: 1308
PII: 1308

Researcher Affiliations

Verney, Mylène
  • Unité PhEED, Laboratoire de Santé Animale, Site de Normandie, ANSES, RD675, 14430, Goustranville, France.
Gautron, Morgane
  • Unité PhEED, Laboratoire de Santé Animale, Site de Normandie, ANSES, RD675, 14430, Goustranville, France.
Lemans, Charlène
  • Unité PhEED, Laboratoire de Santé Animale, Site de Normandie, ANSES, RD675, 14430, Goustranville, France.
Rincé, Alain
  • Unité de Recherche Risques Microbiens U2RM, Normandie-Université, UNICAEN, Caen, France.
Hans, Aymeric
  • Unité PhEED, Laboratoire de Santé Animale, Site de Normandie, ANSES, RD675, 14430, Goustranville, France.
Hébert, Laurent
  • Unité PhEED, Laboratoire de Santé Animale, Site de Normandie, ANSES, RD675, 14430, Goustranville, France. laurent.hebert@anses.fr.

MeSH Terms

  • Animals
  • Antibodies, Protozoan / blood
  • Antibodies, Protozoan / immunology
  • Area Under Curve
  • Enzyme-Linked Immunosorbent Assay / methods
  • Horse Diseases / diagnosis
  • Horse Diseases / parasitology
  • Horses / blood
  • Horses / parasitology
  • Microspheres
  • ROC Curve
  • Recombinant Proteins / immunology
  • Serologic Tests / methods
  • Trypanosoma / immunology
  • Trypanosomiasis / blood
  • Trypanosomiasis / diagnosis
  • Trypanosomiasis / parasitology
  • Trypanosomiasis / veterinary
  • Variant Surface Glycoproteins, Trypanosoma / immunology

Conflict of Interest Statement

The authors declare no competing interests.

References

This article includes 42 references
  1. FAOSTAT. http://www.fao.org/faostat/en/#data/QA/visualize, 2021).
  2. Pritchard JC. Animal traction and transport in the 21st century: getting the priorities right.. Vet J 2010 Dec;186(3):271-4.
    doi: 10.1016/j.tvjl.2010.08.004pubmed: 20833088google scholar: lookup
  3. Burn CC, Dennison TL, Whay HR. Environmental and demographic risk factors for poor welfare in working horses, donkeys and mules in developing countries.. Vet J 2010 Dec;186(3):385-92.
    doi: 10.1016/j.tvjl.2009.09.016pubmed: 19926316google scholar: lookup
  4. OIE. Chapter 7.12. Welfare of working equids. World organisation for Animal Health. Organisation Mondiale de la Santé Animale Paris, pp. 1–10 (2017).
  5. Stringer A. Improving animal health for poverty alleviation and sustainable livelihoods.. Vet Rec 2014 Nov 29;175(21):526-9.
    doi: 10.1136/vr.g6281pubmed: 25431381google scholar: lookup
  6. Büscher P, Gonzatti MI, Hébert L, Inoue N, Pascucci I, Schnaufer A, Suganuma K, Touratier L, Van Reet N. Equine trypanosomosis: enigmas and diagnostic challenges.. Parasit Vectors 2019 May 15;12(1):234.
    doi: 10.1186/s13071-019-3484-xpmc: PMC6518633pubmed: 31092285google scholar: lookup
  7. Barrett MP, Burchmore RJ, Stich A, Lazzari JO, Frasch AC, Cazzulo JJ, Krishna S. The trypanosomiases.. Lancet 2003 Nov 1;362(9394):1469-80.
    doi: 10.1016/S0140-6736(03)14694-6pubmed: 14602444google scholar: lookup
  8. Brun R, Hecker H, Lun ZR. Trypanosoma evansi and T. equiperdum: distribution, biology, treatment and phylogenetic relationship (a review).. Vet Parasitol 1998 Oct;79(2):95-107.
    doi: 10.1016/s0304-4017(98)00146-0pubmed: 9806490google scholar: lookup
  9. Radwanska M, Vereecke N, Deleeuw V, Pinto J, Magez S. Salivarian Trypanosomosis: A Review of Parasites Involved, Their Global Distribution and Their Interaction With the Innate and Adaptive Mammalian Host Immune System.. Front Immunol 2018;9:2253.
    doi: 10.3389/fimmu.2018.02253pmc: PMC6175991pubmed: 30333827google scholar: lookup
  10. Gutierrez C, Desquesnes M, Touratier L, Büscher P. Trypanosoma evansi: recent outbreaks in Europe.. Vet Parasitol 2010 Nov 24;174(1-2):26-9.
    doi: 10.1016/j.vetpar.2010.08.012pubmed: 20851526google scholar: lookup
  11. Pascucci I, Di Provvido A, Cammà C, Di Francesco G, Calistri P, Tittarelli M, Ferri N, Scacchia M, Caporale V. Diagnosis of dourine in outbreaks in Italy.. Vet Parasitol 2013 Mar 31;193(1-3):30-8.
    doi: 10.1016/j.vetpar.2012.12.006pubmed: 23298562google scholar: lookup
  12. Carnes J, Anupama A, Balmer O, Jackson A, Lewis M, Brown R, Cestari I, Desquesnes M, Gendrin C, Hertz-Fowler C, Imamura H, Ivens A, Kořený L, Lai DH, MacLeod A, McDermott SM, Merritt C, Monnerat S, Moon W, Myler P, Phan I, Ramasamy G, Sivam D, Lun ZR, Lukeš J, Stuart K, Schnaufer A. Genome and phylogenetic analyses of Trypanosoma evansi reveal extensive similarity to T. brucei and multiple independent origins for dyskinetoplasty.. PLoS Negl Trop Dis 2015 Jan;9(1):e3404.
  13. Oldrieve G, Verney M, Jaron KS, Hébert L, Matthews KR. Monomorphic Trypanozoon: towards reconciling phylogeny and pathologies.. Microb Genom 2021 Aug;7(8).
    doi: 10.1099/mgen.0.000632pmc: PMC8549356pubmed: 34397347google scholar: lookup
  14. Claes F, Ilgekbayeva GD, Verloo D, Saidouldin TS, Geerts S, Buscher P, Goddeeris BM. Comparison of serological tests for equine trypanosomosis in naturally infected horses from Kazakhstan.. Vet Parasitol 2005 Aug 10;131(3-4):221-5.
    doi: 10.1016/j.vetpar.2005.05.001pubmed: 15951112google scholar: lookup
  15. Zablotskij VT, Georgiu C, de Waal T, Clausen PH, Claes F, Touratier L. The current challenges of dourine: difficulties in differentiating Trypanosoma equiperdum within the subgenus Trypanozoon.. Rev Sci Tech 2003 Dec;22(3):1087-96.
    doi: 10.20506/rst.22.3.1460pubmed: 15005565google scholar: lookup
  16. Kellar KL, Kalwar RR, Dubois KA, Crouse D, Chafin WD, Kane BE. Multiplexed fluorescent bead-based immunoassays for quantitation of human cytokines in serum and culture supernatants.. Cytometry 2001 Sep 1;45(1):27-36.
  17. Vignali DA. Multiplexed particle-based flow cytometric assays.. J Immunol Methods 2000 Sep 21;243(1-2):243-55.
    doi: 10.1016/s0022-1759(00)00238-6pubmed: 10986418google scholar: lookup
  18. Nzou SM, Fujii Y, Miura M, Mwau M, Mwangi AW, Itoh M, Salam MA, Hamano S, Hirayama K, Kaneko S. Development of multiplex serological assay for the detection of human African trypanosomiasis.. Parasitol Int 2016 Apr;65(2):121-7.
    doi: 10.1016/j.parint.2015.10.008pubmed: 26519611google scholar: lookup
  19. Desquesnes M, Bossard G, Patrel D, Herder S, Patout O, Lepetitcolin E, Thevenon S, Berthier D, Pavlovic D, Brugidou R, Jacquiet P, Schelcher F, Faye B, Touratier L, Cuny G. First outbreak of Trypanosoma evansi in camels in metropolitan France.. Vet Rec 2008 Jun 7;162(23):750-2.
    doi: 10.1136/vr.162.23.750pubmed: 18540034google scholar: lookup
  20. Tehseen S, Jahan N, Qamar MF, Desquesnes M, Shahzad MI, Deborggraeve S, Büscher P. Parasitological, serological and molecular survey of Trypanosoma evansi infection in dromedary camels from Cholistan Desert, Pakistan.. Parasit Vectors 2015 Aug 12;8:415.
    doi: 10.1186/s13071-015-1002-3pmc: PMC4532143pubmed: 26259616google scholar: lookup
  21. Ferrer MJ, Wehrendt DP, Bonilla M, Comini MA, Tellez-Iñón MT, Potenza M. Production of Recombinant Trypanosoma cruzi Antigens in Leishmania tarentolae.. Methods Mol Biol 2019;1955:105-118.
    doi: 10.1007/978-1-4939-9148-8_8pubmed: 30868522google scholar: lookup
  22. Rooney B, Piening T, Büscher P, Rogé S, Smales CM. Expression of Trypanosoma brucei gambiense Antigens in Leishmania tarentolae. Potential for Use in Rapid Serodiagnostic Tests (RDTs).. PLoS Negl Trop Dis 2015 Dec;9(12):e0004271.
  23. Birhanu H, Rogé S, Simon T, Baelmans R, Gebrehiwot T, Goddeeris BM, Büscher P. Surra Sero K-SeT, a new immunochromatographic test for serodiagnosis of Trypanosoma evansi infection in domestic animals.. Vet Parasitol 2015 Jul 30;211(3-4):153-7.
    doi: 10.1016/j.vetpar.2015.05.008pubmed: 26012857google scholar: lookup
  24. Rogé S, Van Reet N, Odiwuor S, Tran T, Schildermans K, Vandamme S, Vandenberghe I, Vervecken W, Gillingwater K, Claes F, Devreese B, Guisez Y, Büscher P. Recombinant expression of trypanosome surface glycoproteins in Pichia pastoris for the diagnosis of Trypanosoma evansi infection.. Vet Parasitol 2013 Nov 8;197(3-4):571-9.
    doi: 10.1016/j.vetpar.2013.05.009pubmed: 23747105google scholar: lookup
  25. Boulangé A, Pillay D, Chevtzoff C, Biteau N, Comé de Graça V, Rempeters L, Theodoridis D, Baltz T. Development of a rapid antibody test for point-of-care diagnosis of animal African trypanosomosis.. Vet Parasitol 2017 Jan 15;233:32-38.
    doi: 10.1016/j.vetpar.2016.11.017pubmed: 28043385google scholar: lookup
  26. Davaasuren B, Amgalanbaatar T, Musinguzi SP, Suganuma K, Otgonsuren D, Mossaad E, Narantsatsral S, Battur B, Battsetseg B, Xuan X, Inoue N. The evaluation of GM6-based ELISA and ICT as diagnostic methods on a Mongolian farm with an outbreak of non-tsetse transmitted horse trypanosomosis.. Vet Parasitol 2017 Sep 15;244:123-128.
    doi: 10.1016/j.vetpar.2017.07.036pubmed: 28917303google scholar: lookup
  27. Mizushima D, Amgalanbaatar T, Davaasuren B, Molefe NI, Battur B, Battsetseg B, Inoue N, Yokoyama N, Suganuma K. The utility of an rTeGM6-4r-based immunochromatographic test for the serological diagnosis of non-tsetse-transmitted equine trypanosomosis in rural areas of Mongolia.. Parasitol Res 2018 Sep;117(9):2913-2919.
    doi: 10.1007/s00436-018-5982-8pubmed: 29943319google scholar: lookup
  28. Thuy NT, Goto Y, Lun ZR, Kawazu S, Inoue N. Tandem repeat protein as potential diagnostic antigen for Trypanosoma evansi infection.. Parasitol Res 2012 Feb;110(2):733-9.
    doi: 10.1007/s00436-011-2632-9pubmed: 21927872google scholar: lookup
  29. Ramírez-Iglesias JR, Eleizalde MC, Reyna-Bello A, Mendoza M. Molecular diagnosis of cattle trypanosomes in Venezuela: evidences of Trypanosoma evansi and Trypanosoma vivax infections.. J Parasit Dis 2017 Jun;41(2):450-458.
    doi: 10.1007/s12639-016-0826-xpmc: PMC5447603pubmed: 28615858google scholar: lookup
  30. Balasuriya UB, Shi PY, Wong SJ, Demarest VL, Gardner IA, Hullinger PJ, Ferraro GL, Boone JD, De Cino CL, Glaser AL, Renshaw RW, Ledizet M, Koski RA, MacLachlan NJ. Detection of antibodies to West Nile virus in equine sera using microsphere immunoassay.. J Vet Diagn Invest 2006 Jul;18(4):392-5.
    doi: 10.1177/104063870601800413pubmed: 16921881google scholar: lookup
  31. Beck C, Desprès P, Paulous S, Vanhomwegen J, Lowenski S, Nowotny N, Durand B, Garnier A, Blaise-Boisseau S, Guitton E, Yamanaka T, Zientara S, Lecollinet S. A High-Performance Multiplex Immunoassay for Serodiagnosis of Flavivirus-Associated Neurological Diseases in Horses.. Biomed Res Int 2015;2015:678084.
    doi: 10.1155/2015/678084pmc: PMC4589573pubmed: 26457301google scholar: lookup
  32. Laroucau K, Saqib M, Martin B, Deshayes T, Bertin C, Wernery U, Joseph S, Singha H, Tripathi BN, Beck C. Development of a microsphere-based immunoassay for the serological detection of glanders in equids.. Acta Trop 2020 Jul;207:105463.
  33. Hébert L, Polledo G, Lecouturier F, Giorgi M, Beck C, Lowenski S, Laroucau K, Büscher P, Hans A, Becù T. Serological evidence of equine infectious anaemia, West Nile fever, surra and equine piroplasmosis in a herd of horses in northern Argentina.. Vet Parasitol Reg Stud Reports 2021 Apr;24:100566.
    doi: 10.1016/j.vprsr.2021.100566pubmed: 34024382google scholar: lookup
  34. Hébert L, Guitton E, Madeline A, Géraud T, Carnicer D, Lakhdar L, Pitel PH, Coste M, Laloy E, Giraudet A, Zientara S, Büscher P, Laugier C, Hans A, Petry S, Cauchard J. Validation of a new experimental model for assessing drug efficacy against infection with Trypanosoma equiperdum in horses.. Vet Parasitol 2018 Nov 15;263:27-33.
    doi: 10.1016/j.vetpar.2018.10.005pubmed: 30389021google scholar: lookup
  35. Hébert L, Guitton E, Madeline A, Géraud T, Zientara S, Laugier C, Hans A, Büscher P, Cauchard J, Petry S. Melarsomine hydrochloride (Cymelarsan(®)) fails to cure horses with Trypanosoma equiperdum OVI parasites in their cerebrospinal fluid.. Vet Parasitol 2018 Dec 15;264:47-51.
    doi: 10.1016/j.vetpar.2018.11.005pubmed: 30503091google scholar: lookup
  36. OIE. Terrestrial animal health code. World organisation for Animal Health. Organisation Mondiale de la Santé Animale Paris (2021).
  37. Li Z, Pinto Torres JE, Goossens J, Vertommen D, Caljon G, Sterckx YG, Magez S. An Unbiased Immunization Strategy Results in the Identification of Enolase as a Potential Marker for Nanobody-Based Detection of Trypanosoma evansi.. Vaccines (Basel) 2020 Jul 24;8(3).
    doi: 10.3390/vaccines8030415pmc: PMC7565430pubmed: 32722150google scholar: lookup
  38. Berriman M, Ghedin E, Hertz-Fowler C, Blandin G, Renauld H, Bartholomeu DC, Lennard NJ, Caler E, Hamlin NE, Haas B, Böhme U, Hannick L, Aslett MA, Shallom J, Marcello L, Hou L, Wickstead B, Alsmark UC, Arrowsmith C, Atkin RJ, Barron AJ, Bringaud F, Brooks K, Carrington M, Cherevach I, Chillingworth TJ, Churcher C, Clark LN, Corton CH, Cronin A, Davies RM, Doggett J, Djikeng A, Feldblyum T, Field MC, Fraser A, Goodhead I, Hance Z, Harper D, Harris BR, Hauser H, Hostetler J, Ivens A, Jagels K, Johnson D, Johnson J, Jones K, Kerhornou AX, Koo H, Larke N, Landfear S, Larkin C, Leech V, Line A, Lord A, Macleod A, Mooney PJ, Moule S, Martin DM, Morgan GW, Mungall K, Norbertczak H, Ormond D, Pai G, Peacock CS, Peterson J, Quail MA, Rabbinowitsch E, Rajandream MA, Reitter C, Salzberg SL, Sanders M, Schobel S, Sharp S, Simmonds M, Simpson AJ, Tallon L, Turner CM, Tait A, Tivey AR, Van Aken S, Walker D, Wanless D, Wang S, White B, White O, Whitehead S, Woodward J, Wortman J, Adams MD, Embley TM, Gull K, Ullu E, Barry JD, Fairlamb AH, Opperdoes F, Barrell BG, Donelson JE, Hall N, Fraser CM, Melville SE, El-Sayed NM. The genome of the African trypanosome Trypanosoma brucei.. Science 2005 Jul 15;309(5733):416-22.
    doi: 10.1126/science.1112642pubmed: 16020726google scholar: lookup
  39. Abdille MH, Li SY, Ding J, Suo X. Trypanosoma evansi: Paraflagellar rod protein 1 and 2 are similar but lack common B cell epitopes.. Exp Parasitol 2008 Dec;120(4):411-6.
    doi: 10.1016/j.exppara.2008.08.007pubmed: 18789932google scholar: lookup
  40. Luciani M, Di Febo T, Orsini M, Krasteva I, Cattaneo A, Podaliri Vulpiani M, Di Pancrazio C, Bachi A, Tittarelli M. Trypanosoma equiperdum Low Molecular Weight Proteins As Candidates for Specific Serological Diagnosis of Dourine.. Front Vet Sci 2018;5:40.
    doi: 10.3389/fvets.2018.00040pmc: PMC5844913pubmed: 29556505google scholar: lookup
  41. Verloo D, Holland W, My LN, Thanh NG, Tam PT, Goddeeris B, Vercruysse J, Büscher P. Comparison of serological tests for Trypanosoma evansi natural infections in water buffaloes from north Vietnam.. Vet Parasitol 2000 Sep 20;92(2):87-96.
    doi: 10.1016/s0304-4017(00)00284-3pubmed: 10946132google scholar: lookup
  42. Cuypers B, Van den Broeck F, Van Reet N, Meehan CJ, Cauchard J, Wilkes JM, Claes F, Goddeeris B, Birhanu H, Dujardin JC, Laukens K, Büscher P, Deborggraeve S. Genome-Wide SNP Analysis Reveals Distinct Origins of Trypanosoma evansi and Trypanosoma equiperdum.. Genome Biol Evol 2017 Aug 1;9(8):1990-1997.
    doi: 10.1093/gbe/evx102pmc: PMC5566637pubmed: 28541535google scholar: lookup

Citations

This article has been cited 0 times.