A Novel Running Wheel Mouse Model for Botulism and Its Use for the Evaluation of Postsymptom Antitoxin Efficacy

Abstract

Antitoxin is currently the only approved therapy for botulinum intoxications. The efficacy of antitoxin preparations is evaluated in animals. However, while in practice antitoxin is administered to patients only after symptom onset, in most animal studies, it is tested in relation to time post intoxication. This may be attributed to difficulties in quantitating early botulism symptoms in animals. In the current study, a novel system based on high-resolution monitoring of mouse activity on a running wheel was developed to allow evaluation of post-symptom antitoxin efficacy. The system enables automatic and remote monitoring of 48 mice simultaneously. Based on the nocturnal activity pattern of individual naïve mice, two criteria were defined as the onset of symptoms. Post-symptom treatment with a human-normalized dose of antitoxin was fully protective in mice exposed to 4 LD50 of BoNT/A and BoNT/B. Moreover, for the first time, a high protection rate was obtained in mice treated post-symptomatically, following a challenge with BoNT/E, the fastest acting BoNT. The running wheel system was further modified to develop a mouse model for the evaluation of next-generation therapeutics for progressive botulism at time points where antitoxin is not effective. Exposure of mice to 0.3 LD50 of BoNT/A resulted in long-lasting paralysis and a reduction in running activity for 16-18 days. Antitoxin treatment was no longer effective when administered 72 hr post intoxication, defining the time window to evaluate next-generation therapeutics. Altogether, the running wheel systems presented herein offer quantitative means to evaluate the efficacy of current and future anti-botulinum drugs.

Immunologic and Protective Properties of Subunit- vs. Whole Toxoid-Derived Anti-Botulinum Equine Antitoxin

“…Ten (serotypes A and B) or four (serotype E) hours post-intoxication, animals were treated IM by injection of 215 IU/kg subunit-derived or toxoid-derived (n = 10, each) anti BoNT/A or BoNT/B or 122 IU/kg of anti BoNT/E, to the opposite leg. Animals intoxicated with type E botulinum were treated earlier than those exposed to type A and type B botulinum in agreement with the differences in intoxication kinetics of these serotypes in humans and in animal models of botulism [25,[27][28][29]. For each serotype, a control group (n = 5) was left untreated.…”

“…To evaluate horse anti-H C efficacy, guinea pigs that were exposed to 4 GP IM LD 50 were treated postexposure with either SDA or TDA (n = 20) at a human-normalized antitoxin dose (215 IU/kg for BoNT/A and BoNT/B and 122 IU/kg for BoNT/E, [25]). For BoNT/Aand BoNT/B-exposed animals, the treatment was administered 10 h post-exposure, and for BoNT/E-exposed animals, the treatment was administered 4 h post-exposure.…”

A Rabbit Model for the Evaluation of Drugs for Treating the Chronic Phase of Botulism

“…To date, only a few attempts have been made to develop models for chronic botulism [16,21]. Analogous to severe cases of human botulism, simulating the chronic phase of the disease in animals can theoretically be achieved by extending the survival of intoxicated animals by mechanical ventilation following lethal exposure to BoNT.…”

“…Most recently, our research group developed a running wheel system for detecting botulism symptoms in mice based on voluntary running. Exposure to a sublethal dose of BoNT/A (0.3 LD 50 ) induced a substantial chronic phase (a disease duration of 16-18 days) [16]. In another recent study of mice exposed to sublethal toxin doses, chronic botulism for BoNT/A, BoNT/B, and BoNT/E was developed based on digit abduction scores and moderate-to-severe abdominal paradoxical breathing measurements [21].…”

“…Therefore, at a certain time point, after a critical amount of BoNT molecules are already within neurons, the efficacy of the antitoxin treatment dramatically declines. Indeed, data from studies conducted on animals and from human clinical cases support the notion that there is a critical "therapeutic time window" for effective antitoxin treatment [8,[13][14][15][16][17]. In cases where antitoxin is no longer effective, hospitalization under supportive intensive care and mechanical ventilation may be required.…”