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

Abstract

Botulinum neurotoxins (BoNTs) are the most potent toxins known and they cause the paralytic disease botulism in humans and animals. In order to diagnose botulism, active BoNT must be detected in biological material. Endopep-MS is a sensitive and selective method for serum samples, based on antibody capture, enzymatic cleavage of target peptides, and detection of cleavage products using matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS). In many cases of animal botulism, serum samples are not available or they do not contain detectable amounts of BoNT and liver sampling is an alternative for postmortem examinations. However, the Endopep-MS method is impaired by the inherent protease activity of liver samples. In the presented study, the Endopep-MS method has been successfully modified and validated for analysis of cattle, horse, and avian liver samples, introducing a combination of a salt washing step and a protease inhibitor cocktail. These modifications resulted in a substantial decrease in interfering signals and increase in BoNT-specific signals. This led to a substantial improvement in sensitivity for especially BoNT-C and C/D which are among the most prominent serotypes for animal botulism. Botulism was diagnosed with the new method in liver samples from dead cattle and birds from outbreaks in Sweden.

Recent Developments in Botulinum Neurotoxins Detection

“…The Endopep-MS method has been successfully modified to analyze cattle, horse, and avian liver samples by introducing a salt washing step and a protease inhibitor cocktail. These modifications have resulted in improved sensitivities for BoNT-C and C/D, which are among the most prominent toxinotypes for animal botulism during the diagnosis from outbreaks in Sweden [ 54 ].…”

Single-Domain Antibody Multimers for Detection of Botulinum Neurotoxin Serotypes C, D, and Their Mosaics in Endopep-MS

“…By using BoNT-specific peptide substrates and monitoring the cleavage position by examining the mass of the N-terminal (NT) and C-terminal (CT) cleavage products, the various BoNTs can be differentiated. An immunoaffinity step, with capture antibodies on magnetic beads, prior to incubation with the peptide substrate, is a crucial aspect for detecting and differentiating BoNTs in clinical specimens and culture supernatants [ 11 , 12 , 13 ]. The immunoaffinity not only increases the sensitivity of the test by concentrating BoNT, but it also allows a washing step to remove non-specific proteases that are often present in clinical samples.…”

“…The immunoaffinity not only increases the sensitivity of the test by concentrating BoNT, but it also allows a washing step to remove non-specific proteases that are often present in clinical samples. The sensitivity of Endopep-MS has been shown to equal or exceed that of the mouse bioassay [ 13 , 14 , 15 , 16 , 17 , 18 , 19 ], has shown good performance in an international proficiency test for detection of BoNT/A, B and E [ 20 ], and compares favorably to other in vitro methods for routine diagnostics of clinical specimens and food [ 21 , 22 ].…”

“…While most advances have been made to improve the Endopep-MS assay for BoNT types related to human botulism [ 12 , 14 , 23 ], detection and identification of group III BoNTs related to animal botulism has also progressed [ 13 , 14 , 16 ]. A major challenge is the interference of non-specific proteases that are present in many complex sample matrices, like gastrointestinal contents, liver, feed and environmental samples [ 24 ].…”

Evaluation and Optimization of Microdrop Digital PCR for Detection of Serotype A and B Clostridium botulinum

“…The only currently admissive standard method for detection and identification of botulinum neurotoxin is the mouse bioassays (MBAs) which cause animal ethics issue and are time-consuming (Ferreira et al, 2004 ). At present, the detection methods for toxin-producing species mainly include isolation and culture (CfDCaP (CDC), 2016 ), PCR methods (Cordoba et al, 2001 ; Akbulut and Grant, 2004 ; Heffron and Poxton, 2007 ; Kasai et al, 2007 ; Dahlsten et al, 2008 ; Joshy et al, 2008 ; Fach et al, 2009 ; Hill et al, 2010 ; Kirchner et al, 2010 ; Lindberg et al, 2010 ; Peck et al, 2010 ; Satterfield et al, 2010 ; Anniballi et al, 2013 ; Fohler et al, 2016 ; Le Marechal et al, 2018 ; Masters and Palmer, 2021 ), sequencing (Gonzalez-Escalona et al, 2018 ; Gonzalez-Escalona and Sharma, 2020 ), and matrix-assisted laser desorption ionization-time-of-flight mass spectroscopy (MALDI-TOF MS) based bacterial identification (Kalb et al, 2015 ; Bano et al, 2017 ; Xin et al, 2019 ; Drigo et al, 2020 ; Tevell Aberg et al, 2021 ). Most of these are time-consuming, labor-intensive, and not sensitive enough.…”