Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 9/5/2007
Publication Date: 11/11/2007
Citation: Buhr, R.J., Bourassa, D.V., Cox Jr, N.A., Phillips, R.W., Richardson, L.J., Kelley, L.C. 2007. Detection of botulinum toxin types A, B, E, and F activity using the quail embryo[abstract]. American College of Toxicology Annual. P31, P.73-74.
Technical Abstract: We recently demonstrated an effective new model for the detection of botulinum toxin type A using quail embryos in place of the mouse model. These experiments demonstrated that the Japanese quail embryo at 15 days of incubation was an effective vertebrate animal model to detect the activity of botulinum type A toxin at 1 ng and above. However, there are seven distinct types of botulinum toxin (A-G) of concern for food safety. The objective of this study was to evaluate botulinum toxins types B, E, and F as well as type A using the Japanese quail embryo model. Trypsin activation of the nonproteolytic toxin types B and E is required. Light candling and motion detection were used to confirm viable embryos at 15 days of incubation. The margin of the aircell was marked at the apex and a 2 mm hole was ground through the eggshell. Embryos were injected into the neck/shoulder area with varying toxin concentrations from 10 to 80 ng / 0.05 mL of botulinum toxins. Toxins B and E were mixed with trypsin for 1 hour prior to injection. Following injection, eggs were returned to the incubator and embryo viability was reassessed at 1, 2, and 3 days postinjection. At 3 days postinjection embryos that received 10 ng type A toxin resulted in 80% determined as nonviable, type F toxin 73% were nonviable, while type B and E toxins (without trypsin potentiation) did not differ from control embryos (15-20% nonviable). Subsequent experiments with 10 ng toxin type A resulted in 90% determined as nonviable and 70% nonviable for toxin type F. The noninjected and zero blank control groups had 7-10% nonviable embryos. Embryos injected with 10 ng of type B toxin (trypsin activated) resulted in 40% determined as nonviable and 100% were nonviable at higher concentrations. Embryos injected with 10 ng of type E toxin (trypsin activated) resulted in 90% nonviable, at 20 ng 80% were nonviable, and 100% were nonviable at higher concentrations. The trypsin controls (without toxin) resulted in 20% nonviable embryos at 3 days postinjection. Based on these results, botulinum toxin activity for toxin types A and F and toxin types B and E following trypsin activation are all able to be detected with the Japanese quail model at the minimal level of 10 ng. Index Terms: (for the International Journal of Toxicology) 1. Botulinum toxin types A, B, E, and F 2. Quail embryo assay 3. Trypsin activation of toxin types B and E