The Case of Botulinum Toxin in Milk: Experimental Data

Abstract

Botulinum neurotoxin (BoNT) is the most toxic substance known to man and the causative agent of botulism. Due to its high toxicity and the availability of the producing organism Clostridium botulinum, BoNT is regarded as a potential biological warfare agent. Because of the mild pasteurization process, as well as rapid product distribution and consumption, the milk supply chain has long been considered a potential target of a bioterrorist attack. Since, to our knowledge, no empirical data on the inactivation of BoNT in milk during pasteurization are available at this time, we investigated the activities of BoNT type A (BoNT/A) and BoNT/B, as well as their respective complexes, during a laboratory-scale pasteurization process. When we monitored milk alkaline phosphatase activity, which is an industry-accepted parameter of successfully completed pasteurization, our method proved comparable to the industrial process. After heating raw milk spiked with a set amount of BoNT/A or BoNT/B or one of their respective complexes, the structural integrity of the toxin was determined by enzyme-linked immunosorbent assay (ELISA) and its functional activity by mouse bioassay. We demonstrated that standard pasteurization at 72°C for 15 s inactivates at least 99.99% of BoNT/A and BoNT/B and at least 99.5% of their respective complexes. Our results suggest that if BoNTs or their complexes were deliberately released into the milk supply chain, standard pasteurization conditions would reduce their activity much more dramatically than originally anticipated and thus lower the threat level of the widely discussed "BoNT in milk" scenario.

A reduced graphene oxide-Au based electrochemical biosensor for ultrasensitive detection of enzymatic activity of botulinum neurotoxin A

“…BoNT/A is a protein toxin whose activity is mainly interfered by other protein components in real samples due to non-specific reaction. Since milk is full of proteins, it is always used as complex protein abundant matrix to study protein components interference for BoNT detection [36]. For this purpose, BoNT-LcA protease activity detection was explored in skimmed milk spiked with various concentrations of BoNT-LcA.…”

Complexity of Botulinum Neurotoxins: Challenges for Detection Technology

“…Sharma and colleagues developed an amplified immunoassay based on digoxigenin-labeled pAb to detect the same serotypes in food matrices with assay sensitivities of 60-176 pg/mL (Sharma et al 2006). Even more sensitive and specific immunoassays have been developed with the accessibility of monoclonal antibodies (mAb) (Köhler and Milstein 1975): sandwich ELISAs incorporating mAb resulted in assay sensitivities of around 1-100 pg/mL from culture supernatants, beverages, and complex food matrices (Shone et al 1985;Ferreira et al 1987Ferreira et al , 1990Gibson et al 1987Gibson et al , 1988Ekong et al 1995;Varnum et al 2006;Chiao et al 2008b;Stanker et al 2008;Volland et al 2008;Brooks et al 2010;Scotcher et al 2010;Weingart et al 2010). …”

“…With respect to the detection of active versus inactive BoNT, it should be noted that only very few ELISA formats based on mAb have been shown to be able to discriminate the active toxin from its denatured form (Weingart et al 2010). This might be relevant in thermal inactivation studies when comparing ELISA results with activity-based results.…”

Anaerobic bacteria as producers of antibiotics

“…Some pathogenic clostridia are infamous for their ability to produce peptide toxins such as tetanus and botulinum toxin (Hatheway 1990;Weingart et al 2010), whereas nonpathogenic Clostridium spp. play eminent roles in the bioremediation of complex organic compounds and organic solvent production ( Fig.…”