In vitro production of Trypanosoma equiperdum antigen and its evaluation for use in serodiagnosis of dourine.
Abstract: A modified Baltz's in vitro cultivation system for the propagation of Trypanosoma equiperdum strain OVI was established to develop a replacement for the conventional production procedure of dourine diagnostic antigen in rats. To increase trypanosome yields we designed an optimized culture medium by addition of supplemental compounds. Trypanosomes were adapted to this medium by two succeeding cultivation steps which led to a substantial proliferation rate and an increased cell density tolerance, respectively. As a result, adapted parasites could be propagated to maximum cell densities of >2×10(6) cells/ml, facilitating in vitro antigen production in preparative quantities comparable to the conventional method. A panel of 180 horse field sera, previously sent for testing to the German National Reference Laboratory for Dourine, was tested by complement fixation test using culture-derived as well as conventionally produced dourine antigen. Cohen's kappa values for results obtained with two batches of culture-derived antigen as compared to conventional antigen were 0.91 (95% confidence interval [CI]: 82.2-99.7) and 0.83 (95% CI: 70.3-95.3), respectively. Performance of antigens for diagnostic purposes was characterized in an inter-laboratory comparative study deploying 14 sera from horses with defined dourine statuses. Complement fixation test results from 15 participating European laboratories showed a diagnostic sensitivity of 94.1% (95% CI: 89.4-98.7) and a diagnostic specificity of 96.2% (95% CI: 92.5-99.9) for conventional antigen and a slightly higher diagnostic sensitivity of 96.0% (95% CI: 92.2-99.8) and a diagnostic specificity of 97.1% (95% CI: 94.0-100) for culture-derived antigen. We conclude that our novel approach for dourine antigen production from in vitro-grown trypanosomes described and evaluated herein meets the requirements for the prospective purpose in quantitative and qualitative terms and should be considered by the competent authorities as an alternative for the animal experiment currently prescribed by international standards.
Copyright © 2016. Published by Elsevier B.V.
Publication Date: 2016-04-23 PubMed ID: 27198790DOI: 10.1016/j.vetpar.2016.04.032Google Scholar: Lookup
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Summary
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This study presents a new methodology for producing dourine diagnostic antigens through the cultivation of the Trypanosoma equiperdum strain in vitro. The team tested their substance using a panel of horsefield sera and concluded that the new approach showed promising diagnositc sensitivity and specificity for dourine.
Scientific Background
- This research was conducted in response to the need for a more efficient and ethical method of producing diagnostic antigens for dourine, a sexually transmitted disease in horses caused by Trypanosoma equiperdum.
- Previously, these antigens were conventionally produced in rats, a procedure which this study aims to replace with an in vitro propagation system.
Modified Baltz’s in vitro Cultivation System
- The researchers adopted a modified version of the Baltz’s in vitro cultivation system to propagate Trypanosoma equiperdum.
- An optimized culture medium was created. This, alongside a two-step adaptation process, resulted in a higher rate of cell proliferation and cell density tolerance. Consequently, the culture-derived antigen could be grown in large quantities for diagnostic use.
Diagnostic Evaluation and Comparative Inter-laboratory Study
- A panel of 180 horse field sera was tested using both culture-derived and conventionally produced dourine antigens. High Cohen’s kappa values were observed, indicating substantial correlation in the diagnostic results with both types of antigen.
- An inter-laboratory comparative study was performed amongst 15 European labs. This entailed testing 14 sera from horses with defined dourine statuses.
Diagnostic Sensitivity and Specificity of the Culture-derived Antigen
- The culture-derived antigen delivered a diagnostic sensitivity of 96.0% and a diagnostic specificity of 97.1%. This, the researchers contend, was slightly superior to the performance of the conventional antigen.
Conclusion
- The study concludes that the novel in vitro culture method for dourine antigen production is both quantitatively and qualitatively reliable.
- The research team recommends its adoption as an alternative to traditional animal experiments, subject to approval by the relevant authorities.
Cite This Article
APA
Bassarak B, Moser I, Menge C.
(2016).
In vitro production of Trypanosoma equiperdum antigen and its evaluation for use in serodiagnosis of dourine.
Vet Parasitol, 223, 133-140.
https://doi.org/10.1016/j.vetpar.2016.04.032 Publication
Researcher Affiliations
- Friedrich-Loeffler-Institut/Federal Research Institute for Animal Health, Institute of Molecular Pathogenesis, Naumburger Straße 96a, 07743 Jena, Germany; German National Reference Laboratory for Dourine, Friedrich-Loeffler-Institut/Federal Research Institute for Animal Health, Institute of Molecular Pathogenesis, Naumburger Straße 96a, 07743 Jena, Germany. Electronic address: mail@bassarak.de.
- Friedrich-Loeffler-Institut/Federal Research Institute for Animal Health, Institute of Molecular Pathogenesis, Naumburger Straße 96a, 07743 Jena, Germany; German National Reference Laboratory for Dourine, Friedrich-Loeffler-Institut/Federal Research Institute for Animal Health, Institute of Molecular Pathogenesis, Naumburger Straße 96a, 07743 Jena, Germany. Electronic address: irmgard.moser@fli.bund.de.
- Friedrich-Loeffler-Institut/Federal Research Institute for Animal Health, Institute of Molecular Pathogenesis, Naumburger Straße 96a, 07743 Jena, Germany. Electronic address: christian.menge@fli.bund.de.
MeSH Terms
- Animals
- Antigens, Protozoan / isolation & purification
- Dourine / diagnosis
- Horse Diseases / parasitology
- Horses
- Male
- Rats
- Rats, Wistar
- Serologic Tests / methods
- Serologic Tests / veterinary
- Trypanosoma / classification
- Trypanosoma / metabolism
Citations
This article has been cited 2 times.- 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.
- Suganuma K, Yamasaki S, Molefe NI, Musinguzi PS, Davaasuren B, Mossaad E, Narantsatsral S, Battur B, Battsetseg B, Inoue N. The establishment of in vitro culture and drug screening systems for a newly isolated strain of Trypanosoma equiperdum.. Int J Parasitol Drugs Drug Resist 2017 Aug;7(2):200-205.
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