**3. The mouse bioassay**

In the laboratory, a rodent bioassay is considered the "gold standard" method for detecting BoNTs [8–10]. Despite much effort to replace the use of animals, it is still the most sensitive and reliable assay to model all aspects of BoNT intoxication: binding, translocation, enzymatic activity, and pathology. Alternatives to the mouse bioassay have been developed (discussed below) with shorter assay times and equal or greater sensitivity.

The mouse assay quantitatively determines the amount of BoNT required to kill all mice in a test group. This measurement is expressed as a minimal lethal dose (MLD). Although protocols may vary, mice are usually injected intraperitoneally with 0.5 mL of BoNT sample in a dilution series and then monitored over several days for signs of intoxication and death

[11, 12]. If enough sample is available for an assay, the specific neurotoxin can be identi‐ fied using neutralization with antibodies specific to each of the neurotoxin serotypes (A– G). The toxin serotype is therefore revealed based on which antibody confers protection from death. The mouse bioassay is highly sensitive and useful for detection of different neurotox‐ ins in different matrices. However, despite its versatility, the mouse assay has limitations that include: long assay times and the use of animals requiring specialized animal facilities, substantial costs, trained staff, and consideration of ethical issues, especially when death is used as an endpoint. There is also substantial variation in results observed among different research laboratories [8–12].

Alternative "refined" animal assays that do not use death as an endpoint, such as the mouse phrenic nerve hemi-diaphragm assay, have been evaluated [13, 14]. Despite being more sensitive and rapid compared to the use of whole animals, these assays usually require the use of specific equipment and personnel with specialized training. Furthermore, these alternative animal assays are not feasible with larger samples and those containing a complex matrix. However, a recent study described an *in vivo* assay using a toe-spread reflex model. This method was used to detect neurotoxins in simple buffer solutions, samples containing serum, and milk [15]. This new assay provides results more quickly than standard mouse bioassays. Whether these results can be translated into a user-friendly, deployable kit has yet to be determined.
