Advancing botulism diagnostics: integrating molecularly specific in vitro platforms and high-sensitivity analytical technologies as alternatives to the mouse bioassay

Authors

DOI:

https://doi.org/10.14739/2310-1210.2026.3.344174

Keywords:

botulinum toxins, botulism, diagnostic techniques and procedures, mass spectrometry, endopeptidases, SNARE proteins, in vitro techniques

Abstract

Accurate detection of botulinum neurotoxin (BoNT) requires assessing both its proteolytic SNARE-cleaving activity and its ability to inhibit synaptic transmission. While the mouse bioassay remains the traditional diagnostic standard, its prolonged turnaround time, limited reproducibility, and ethical constraints underscore the urgent need for advanced in vitro methodologies. Recent advances in analytical chemistry, stem-cell-derived neuronal platforms, and multi-omics profiling enable the development of sensitive systems that replicate BoNT’s molecular and functional mechanisms with greater precision than conventional in vivo models.

Aim. To evaluate current methodologies for the diagnosis and functional analysis of botulinum neurotoxin and, synthesizing these findings, to propose a conceptual algorithmic model for comprehensive neurotoxin detection independent of in vivo methods.

Materials and methods. A literature review was conducted utilizing the PubMed, Scopus, and Web of Science databases. Studies detailing molecular, cellular, multi-omics, and computational approaches to BoNT detection were included. The extracted data were synthesized across molecular, cellular, and systems-level domains to construct an integrated diagnostic model.

Results. Modern diagnostic strategies increasingly surpass the mouse bioassay in terms of sensitivity, specificity, and mechanistic insight. SNARE-specific proteolytic profiling, notably Endopep-MS, enables precise detection of BoNT serotypes and their functional states by quantifying cleavage kinetics across a diverse array of substrates. Complementary human iPSC-derived neuronal platforms allow for direct functional assessment, revealing electrophysiological suppression, altered calcium signaling, and reporter-confirmed SNARE cleavage within living cells. Multi-omics analyses, including single-cell transcriptomics, epigenomics, and metabolomics, capture early stress signatures, serotype-specific responses, and determinants of neuronal susceptibility. Additional resolution is provided by receptor-binding kinetics and intracellular trafficking studies, which elucidate how serotype-dependent variations dictate overall toxicity. The integration of these molecular, cellular, and systems-level insights establishes the foundation for the ULTIMA-BoNT framework, a unified platform designed for high-precision BoNT detection.

Conclusions. The convergence of proteolytic assays, physiologically relevant neuronal models, and multi-omics analytics presents a robust, reproducible, and ethically sustainable alternative to the mouse bioassay. This integrative approach not only provides profound mechanistic insights but also supports the predictive analysis of emerging BoNT variants.

Author Biographies

Ya. D. Bondarenko, Kharkiv National Medical University

Student, Third Faculty of Medicine; Medical Faculty, Friedrich-Alexander University of Erlangen-Nuremberg, Germany

O. V. Kochnieva, Kharkiv National Medical University

PhD, Associate Professor of the Department of Microbiology, Virology and Immunology named after D. P. Grynyov

References

  1. Drigo I, Tonon E, Pascoletti S, Anniballi F, Kalb SR, Bano L. Detection of active BoNT/C and D by EndoPep-MS using MALDI Biotyper instrument and comparison with the mouse test bioassay. Toxins (Basel). 2020;13(1):10. doi: https://doi.org/10.3390/toxins13010010
    | |
  2. Tevell Åberg A, Karlsson I, Hedeland M. Modification and validation of the Endopep-mass spectrometry method for botulinum neurotoxin detection in liver samples with application to samples collected during animal botulism outbreaks. Anal Bioanal Chem. 2021;413(2):345-54. doi: https://doi.org/10.1007/s00216-020-03001-z
    | |
  3. Pellett S, Tepp WH, Johnson EA. Critical analysis of neuronal cell and the mouse bioassay for detection of botulinum neurotoxins. Toxins. 2019;11(12):713. doi: https://doi.org/10.3390/toxins11120713
    | |
  4. Gray B, Cadd V, Elliott M, Beard M. The in vitro detection of botulinum neurotoxin-cleaved endogenous VAMP is epitope-dependent. Toxicol In Vitro. 2018;48:255-61. doi: https://doi.org/10.1016/j.tiv.2018.01.016
    | |
  5. Pellett S. Progress in cell-based assays for botulinum neurotoxin detection. Curr Top Microbiol Immunol. 2013;364:257-85. doi: https://doi.org/10.1007/978-3-642-33570-9_12 23239357
    | |
  6. Steinberg M, Wilk LV, Stern D, Weisemann J, Messelhäußer U, Wittwer M, et al. Validation of an Endopep-suspension immunoassay for the diagnostics of human botulism. ALTEX. 2025;42(4):636-56. doi: https://doi.org/10.14573/altex.2412181
    | |
  7. Hoyt KM, Barr JR, Hopkins AO, Dykes JK, Lúquez C, Kalb SR. Validation of a clinical assay for botulinum neurotoxins through mass spectrometric detection. J Clin Microbiol. 2024;62(6):e0162923. doi: https://doi.org/10.1128/jcm.01629-23
    | |
  8. Wang D, Baudys J, Ye Y, Rees JC, Barr JR, Pirkle JL, et al. Improved detection of botulinum neurotoxin serotype A by Endopep-MS through peptide substrate modification. Anal Biochem. 2013;432(2):115-23. doi: https://doi.org/10.1016/j.ab.2012.09.021
    | |
  9. Barr JR, Moura H, Boyer AE, Woolfitt AR, Kalb SR, Pavlopoulos A, et al. Botulinum neurotoxin detection and differentiation by mass spectrometry. Emerg Infect Dis. 2005;11(10):1578-83. doi: https://doi.org/10.3201/eid1110.041279
    | |
  10. Chen S, Barbieri JT. Association of botulinum neurotoxin serotype A light chain with plasma membrane-bound SNAP-25. J Biol Chem. 2011;286(17):15067-72. doi: https://doi.org/10.1074/jbc.M111.224493
    | |
  11. Chen S, Hall C, Barbieri JT. Substrate recognition of VAMP-2 by botulinum neurotoxin B and tetanus neurotoxin. J Biol Chem. 2008;283(30):21153-9. doi: https://doi.org/10.1074/jbc.M800611200
    | |
  12. Hallis B, James BA, Shone CC. Development of novel assays for botulinum type A and B neurotoxins based on their endopeptidase activities. J Clin Microbiol. 1996;34(8):1934-8. doi: https://doi.org/10.1128/jcm.34.8.1934-1938.1996
    | |
  13. Michalik M, Grzybowski J, Ligieza J, Reiss J. Enzyme-linked immunosorbent assay (ELISA) for detection and differentiation of Clostridium botulinum toxins type A and B. J Immunol Methods. 1986;93(2):225-30. doi: https://doi.org/10.1016/0022-1759(86)90193-6
    | |
  14. Simon S, Fiebig U, Liu Y, Tierney R, Dano J, Worbs S, et al. Recommended immunological strategies to screen for botulinum neurotoxin-containing samples. Toxins (Basel). 2015;7(12):5011-34. doi: https://doi.org/10.3390/toxins7124860
    | |
  15. Chiao DJ, Shyu RH, Hu CS, Chiang HY, Tang SS. Colloidal gold-based immunochromatographic assay for detection of botulinum neurotoxin type B. J Chromatogr B Analyt Technol Biomed Life Sci. 2004;809(1):37-41. doi: https://doi.org/10.1016/j.jchromb.2004.05.033
    | |
  16. De Medici D, Anniballi F, Wyatt GM, Lindström M, Messelhäusser U, Aldus CF, et al. Multiplex PCR for detection of botulinum neurotoxin-producing clostridia in clinical, food, and environmental samples. Appl Environ Microbiol. 2009;75(20):6457-61. doi: https://doi.org/10.1128/AEM.00805-09
    | |
  17. Hill BJ, Skerry JC, Smith TJ, Arnon SS, Douek DC. Universal and specific quantitative detection of botulinum neurotoxin genes. BMC Microbiol. 2010;10:267. doi: https://doi.org/10.1186/1471-2180-10-267
    | |
  18. Lövenklev M, Holst E, Borch E, Rådström P. Relative neurotoxin gene expression in Clostridium botulinum type B determined using quantitative reverse transcription-PCR. Appl Environ Microbiol. 2004;70(5):2919-27. doi: https://doi.org/10.1128/AEM.70.5.2919-2927.2004
    | |
  19. Franciosa G, Hatheway CL, Aureli P. Detection of a deletion in the type B neurotoxin gene of Clostridium botulinum A(B) strains by a two-step PCR. Lett Appl Microbiol. 1998;26(6):442-6. doi: https://doi.org/10.1046/j.1472-765x.1998.00367.x
    | |
  20. Lindström M, Korkeala H. Laboratory diagnostics of botulism. Clin Microbiol Rev. 2006;19(2):298-314. doi: https://doi.org/10.1128/CMR.19.2.298-314.2006
    | |
  21. Rasooly R, Do PM. Development of an in vitro activity assay as an alternative to the mouse bioassay for Clostridium botulinum neurotoxin type A. Appl Environ Microbiol. 2008;74(14):4309-13. doi: https://doi.org/10.1128/AEM.00617-08
    | |
  22. Zechmeister TC, Farnleitner AH, Rocke TE, Pittner F, Rosengarten R, Mach RL, et al. PCR und ELISA – Alternativen zum Maustest für die Analyse des Botulismus-Neurotoxin-C1-Giftbildungspotentiales in Umweltproben? ALTEX. 2002;19 Suppl 1:49-54.
  23. Kalb SR, Krilich JC, Dykes JK, Lúquez C, Maslanka SE, Barr JR. Detection of botulinum toxins A, B, E, and F in foods by Endopep-MS. J Agric Food Chem. 2015;63(4):1133-41. doi: https://doi.org/10.1021/jf505482b
    | |
  24. Pellett S, Tepp WH, Bradshaw M, Kalb SR, Dykes JK, Lin G, et al. Purification and characterization of botulinum neurotoxin FA from a genetically modified Clostridium botulinum strain. mSphere. 2016;1(1):e00100-15. doi: https://doi.org/10.1128/mSphere.00100-15
    | |
  25. Rosen O, Feldberg L, Yamin TS, Dor E, Barnea A, Weissberg A, et al. Development of a multiplex Endopep-MS assay for simultaneous detection of botulinum toxins A, B and E. Sci Rep. 2017;7(1):14859. doi: https://doi.org/10.1038/s41598-017-14911-x
    | |
  26. Poras H, Ouimet T, Orng SV, Fournié-Zaluski MC, Popoff MR, Roques BP. Detection and quantification of botulinum neurotoxin type A by a novel rapid in vitro fluorimetric assay. Appl Environ Microbiol. 2009;75(13):4382-90. doi: https://doi.org/10.1128/AEM.00091-09
    | |
  27. Björnstad K, Tevell Åberg A, Kalb SR, Wang D, Barr JR, Bondesson U, et al. Validation of the Endopep-MS method for qualitative detection of active botulinum neurotoxins in human and chicken serum. Anal Bioanal Chem. 2014;406(28):7149-61. doi: https://doi.org/10.1007/s00216-014-8170-4
    | |
  28. Wang D, Krilich J, Baudys J, Barr JR, Kalb SR. Optimization of peptide substrates for botulinum neurotoxin E improves detection sensitivity in the Endopep-MS assay. Anal Biochem. 2015;468:15-21. doi: https://doi.org/10.1016/j.ab.2014.08.026
    | |
  29. Lamotte JD, Roqueviere S, Gautier H, Raban E, Bouré C, Fonfria E, et al. hiPSC-derived neurons provide a robust and physiologically relevant in vitro platform to test botulinum neurotoxins. Front Pharmacol. 2021;11:617867. doi: https://doi.org/10.3389/fphar.2020.617867
    | |
  30. Lin CY, Yoshida M, Li LT, Ikenaka A, Oshima S, Nakagawa K, et al. iPSC-derived functional human neuromuscular junctions model the pathophysiology of neuromuscular diseases. JCI Insight. 2019;4(18):e124299. doi: https://doi.org/10.1172/jci.insight.124299
    | |
  31. Vila OF, Qu Y, Vunjak-Novakovic G. In vitro models of neuromuscular junctions and their potential for novel drug discovery and development. Expert Opin Drug Discov. 2020;15(3):307-17. doi: https://doi.org/10.1080/17460441.2020.1700225
    | |
  32. de Lamotte JD, Polentes J, Roussange F, Lesueur L, Feurgard P, Perrier A, et al. Optogenetically controlled human functional motor endplate for testing botulinum neurotoxins. Stem Cell Res Ther. 2021;12(1):599. doi: https://doi.org/10.1186/s13287-021-02665-3
    | |
  33. von Berg L, Stern D, Weisemann J, Rummel A, Dorner MB, Dorner BG. Optimization of SNAP-25 and VAMP-2 cleavage by botulinum neurotoxin serotypes A–F employing Taguchi design-of-experiments. Toxins (Basel). 2019;11(10):588. doi: https://doi.org/10.3390/toxins11100588
    | |
  34. Cotter L, Yu F, Roqueviere S, Duchesne de Lamotte J, Krupp J, Dong M, Nicoleau C. Split luciferase-based assay to detect botulinum neurotoxins using hiPSC-derived motor neurons. Commun Biol. 2023;6(1):122. doi: https://doi.org/10.1038/s42003-023-04495-w
    | |
  35. Dong M, Tepp WH, Johnson EA, Chapman ER. Using fluorescent sensors to detect botulinum neurotoxin activity in vitro and in living cells. Proc Natl Acad Sci U S A. 2004;101(41):14701-6. doi: https://doi.org/10.1073/pnas.0404107101
    | |
  36. Stuart T, Butler A, Hoffman P, Hafemeister C, Papalexi E, Mauck WM 3rd, et al. Comprehensive Integration of Single-Cell Data. Cell. 2019;177(7):1888-902.e21. doi: https://doi.org/10.1016/j.cell.2019.05.031
    | |
  37. Mueller K, Saha K. Single Cell Technologies to Dissect Heterogenous Immune Cell Therapy Products. Curr Opin Biomed Eng. 2021;20:100343. doi: https://doi.org/10.1016/j.cobme.2021.100343
    | |
  38. Wu X, Yang X, Dai Y, Zhao Z, Zhu J, Guo H, Yang R. Single-cell sequencing to multi-omics: technologies and applications. Biomark Res. 2024;12(1):110. doi: https://doi.org/10.1186/s40364-024-00643-4
    | |
  39. Hetz C, Saxena S. ER stress and the unfolded protein response in neurodegeneration. Nat Rev Neurol. 2017;13(8):477-91. doi: https://doi.org/10.1038/nrneurol.2017.99
    | |
  40. Berliocchi L, Fava E, Leist M, Horvat V, Dinsdale D, Read D, Nicotera P. Botulinum neurotoxin C initiates two different programs for neurite degeneration and neuronal apoptosis. J Cell Biol. 2005;168(4):607-18. doi: https://doi.org/10.1083/jcb.200406126
    | |
  41. Otte AP, Kwaks TH. Gene repression by Polycomb group protein complexes: a distinct complex for every occasion? Curr Opin Genet Dev. 2003;13(5):448-54. doi: https://doi.org/10.1016/s0959-437x(03)00108-4
    | |
  42. Flores A, Ramirez-Franco J, Desplantes R, Debreux K, Ferracci G, Wernert F, et al. Gangliosides interact with synaptotagmin to form the high-affinity receptor complex for botulinum neurotoxin B. Proc Natl Acad Sci U S A. 2019;116(36):18098-108. doi: https://doi.org/10.1073/pnas.1908051116
    | |
  43. Yowler BC, Schengrund CL. Botulinum neurotoxin A changes conformation upon binding to ganglioside GT1b. Biochemistry. 2004;43(30):9725-31. doi: https://doi.org/10.1021/bi0494673
    | |
  44. Weisemann J, Stern D, Mahrhold S, Dorner BG, Rummel A. Botulinum Neurotoxin Serotype A Recognizes Its Protein Receptor SV2 by a Different Mechanism than Botulinum Neurotoxin B Synaptotagmin. Toxins (Basel). 2016;8(5):154. doi: https://doi.org/10.3390/toxins8050154
    | |
  45. Dong M, Yeh F, Tepp WH, Dean C, Johnson EA, Janz R, et al. SV2 is the protein receptor for botulinum neurotoxin A. Science. 2006;312(5773):592-6. doi: https://doi.org/10.1126/science.1123654
    | |
  46. Lúquez C, Raphael BH, Maslanka SE. Neurotoxin gene clusters in Clostridium botulinum type Ab strains. Appl Environ Microbiol. 2009;75(19):6094-101. doi: https://doi.org/10.1128/AEM.01009-09
    | |
  47. Dineen SS, Bradshaw M, Johnson EA. Neurotoxin gene clusters in Clostridium botulinum type A strains: sequence comparison and evolutionary implications. Curr Microbiol. 2003;46(5):345-52. doi: https://doi.org/10.1007/s00284-002-3851-1
    | |
  48. Carter AT, Austin JW, Weedmark KA, Peck MW. Evolution of Chromosomal Clostridium botulinum Type E Neurotoxin Gene Clusters: Evidence Provided by Their Rare Plasmid-Borne Counterparts. Genome Biol Evol. 2016;8(3):540-55. doi: https://doi.org/10.1093/gbe/evw017
    | |

Downloads

Additional Files

Published

2026-06-11

How to Cite

1.
Bondarenko YD, Kochnieva OV. Advancing botulism diagnostics: integrating molecularly specific in vitro platforms and high-sensitivity analytical technologies as alternatives to the mouse bioassay. Zaporozhye Medical Journal [Internet]. 2026Jun.11 [cited 2026Jun.11];28(3):267-73. Available from: https://zmj.zsmu.edu.ua/article/view/344174

Issue

Vol. 28 No. 3 (2026): Zaporozhye Medical Journal

Section

Review

License

Copyright (c) 2026 Ya. D. Bondarenko, O. V. Kochnieva

Creative Commons License

This work is licensed under a Creative Commons Attribution 4.0 International License.

Authors who publish with this journal agree to the following terms:

Authors retain copyright and grant the journal right of first publication with the work simultaneously licensed under a Creative Commons Attribution License that allows others to share the work with an acknowledgement of the work's authorship and initial publication in this journal.  Лицензия Creative Commons