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United States Department of Agriculture

Agricultural Research Service

Research Project: INTERVENTIONS AND METHODOLOGIES TO REDUCE HUMAN FOOD-BORNE BACTERIAL PATHOGENS IN CHICKENS Title: Development of a quail embryo model for the detection of botulinum neurotoxin activity

Authors
item Buhr, Richard
item Bourassa, Dianna
item Cox, Nelson
item Richardson, Larry
item Phillips, R - FSIS
item Kelley, L - FSIS

Submitted to: Poultry Science
Publication Type: Abstract Only
Publication Acceptance Date: October 10, 2008
Publication Date: January 26, 2009
Citation: Buhr, R.J., Bourassa, D.V., Cox Jr, N.A., Richardson, L.J., Phillips, R.W., Kelley, L.C. 2009. Development of a quail embryo model for the detection of botulinum neurotoxin activity. Poultry Science. 88(Suppl 1):M75. P.24.

Technical Abstract: Clostridium botulinum is a ubiquitous microorganism that under anaerobic conditions produces botulinum neurotoxins. In regards to both food-borne illness and the potential use of botulinum toxin as a biological weapon, the capability to assess the amount of toxin in a food or environmental sample efficiently is critical. Currently the mouse toxicity and neutralization assay is used for assessment of botulinum toxin activity and can detect a little as 20 pg/mL. However, there are safety concerns for handling needles and moving animals with samples containing undetermined amounts of toxin. In addition, there is growing pressure to replace the mouse LD50 assay for ethical concerns over the use of death by asphyxiation of the mice as the test endpoint. The objective of this study was to develop and evaluate a screening assay for detecting biologically active botulinum neurotoxins using Japanese quail embryos. Quail embryos at day 15 of incubation were injected into the neck/shoulder area with botulinum toxin type A (0.05 to 250 ng / 0.05 mL) and types B, E, and F (10 to 80 ng / 0.05 mL). At 3 days post-injection, embryos injected at 0.1 ng or higher with type A toxin had significantly more embryos than the control determined to be non-viable (21.5 vs. 2.5%). Neurotoxins B, E, and F were all detected at 10 ng/embryo with 40, 90, and 70% of embryos determined to be non-viable. Premixing of the toxin type A with type A specific antibody demonstrated that the depression in the ability of the quail embryos to pip and hatch was indeed attributable to biologically active toxin (80 vs. 14%). These experiments demonstrate that the Japanese quail embryo is an effective vertebrate animal model to detect the biological activity of botulinum neurotoxins A, B, E, and F. The minimal detectable dosage by the quail embryos is 100 pg of toxin (14 µg/kg of body weight). Utilization of a quail embryo may be a beneficial model for the analysis of botulinum toxins activity by enhancing personnel safety and since BoNTs does not kill the quail embryo, but restrict the embryo’s ability to progress to pipping into the aircell and through the eggshell, non-viable embryos can be presumptively euthanized upon detection.

Last Modified: 10/20/2014
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