Open Access An Alternative In Vivo Method to Refine the Mouse Bioassay for Botulinum Toxin Detection

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Abstract:

Botulism is a rare, life-threatening paralytic disease of both humans and animals that is caused by botulinum neurotoxins (BoNT). Botulism is confirmed in the laboratory by the detection of BoNT in clinical specimens, contaminated foods, and cultures. Despite efforts to develop an in vitro method for botulinum toxin detection, the mouse bioassay remains the standard test for laboratory confirmation of this disease. In this study, we evaluated the use of a nonlethal mouse toe-spread reflex model to detect BoNT spiked into buffer, serum, and milk samples. Samples spiked with toxin serotype A and nontoxin control samples were injected into the left and right extensor digitorum longus muscles, respectively. Digital photographs at 0,8, and 24 h were used to obtain objective measurements through effective paralysis scores, which were determined by comparing the width-to-length ratio between right and left feet. Both objective measurements and clinical observation could accurately identify over 80% of animals injected with 1 LD50 (4.3 pg) BoNT type A within 24 h. Half of animals injected with 0.5 LD50 BoNT type A and none injected with 0.25 LD50 demonstrated localized paralysis. Preincubating the toxin with antitoxin prevented the development of positive effective paralysis scores, demonstrating that (1) the effect was specific for BoNT and (2) identification of toxin serotype could be achieved by using this method. These results suggest that the mouse toe-spread reflex model may be a more humane alternative to the current mouse bioassay for laboratory investigations of botulism.

Document Type: Research Article

Affiliations: 1: Division of Scientific Resources, National Center for Emerging and Zoonotic Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, USA 2: Division of Foodborne, Waterborne and Environmental Diseases, National Center for Emerging and Zoonotic Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, USA 3: Medical Research Institute of Chemical Defense, United States Army, Aberdeen Proving Ground, Maryland, USA 4: Division of Foodborne, Waterborne and Environmental Diseases, National Center for Emerging and Zoonotic Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, USA. SMaslanka@cdc.gov

Publication date: June 1, 2011

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  • Comparative Medicine (CM), an international journal of comparative and experimental medicine, is the leading English-language publication in the field and is ranked by the Science Citation Index in the upper third of all scientific journals. The mission of CM is to disseminate high-quality, peer-reviewed information that expands biomedical knowledge and promotes human and animal health through the study of laboratory animal disease, animal models of disease, and basic biologic mechanisms related to disease in people and animals.

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