Submitted to: American Association of Veterinary Laboratory Diagnosticians
Publication Type: Abstract only
Publication Acceptance Date: 7/30/2007
Publication Date: 10/17/2007
Citation: Buhr, R.J., Cox Jr, N.A., Richardson, L.J., Phillips, R.W., Kelley, L.C. 2007. Development of a quail embryo model for the detection of botulinum toxin type A activity. American Association of Veterinary Laboratory Diagnosticians. P.112. Interpretive Summary:
Technical Abstract: Clostridium botulinum is a ubiquitous microorganism which under certain anaerobic conditions can produce botulinum toxins. Due to concerns 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 assay is used as the model for assessment of botulinum toxin activity. However, the mouse model requires 36 hours for completion of the assay. In addition there are safety concerns for handling needles and moving animals with samples containing undetermined amounts of toxin. Mouse colonies are also expensive to maintain. The objective of the study was to develop and evaluate a new screening assay for determination of biologically active botulinum toxin utilizing incubating quail embryos (Coturnix coturnix japonica,). Fertile quail eggs were incubated for 15 days under standard conditions and embryo viability was assessed by both light candling and with motion detection (Digital Egg Monitor). Eggs with viable embryos were marked at the margin of the air cell closest to the blunt end of the egg, and a 2 mm hole was ground through the eggshell. The embryos were then injected into the neck/shoulder area with varying concentrations (1 to 250 ng / 0.05 mL) of botulinum toxin type A. Following injection, eggs were returned to the incubator and embryo viability was reassessed at 1, 2, and 3 days post-injection. At 1 day post-injection, embryos injected at 20 ng or higher with type A toxin had more than 75% of the embryos determined to be not viable. The 5 and 10 ng injection doses had 53% and 50%, respectively, of the embryos remained viable at 1 day post-injection. After 3 days post-injection both the 5 and 10 ng injection dosages had less than 50% viability, the 1 ng injection dose had 73% viable embryos, while the control group (0 ng of toxin) retained a high viability at 93%. Premixing of the toxin for 1 hour with serotype A specific antibody (in excess) demonstrated that the depression in the ability of the quail embryos to pip and hatch was indeed attributable to biologically active type A toxin. Embryos injected with toxin dosages from 1 to 50 ng that were preincubated with serotype A specific antibody maintained a viability of greater than 80% after 3 days post-injection compared to less than 14% viability for those embryos injected at the same dosages without preincubation of the toxin with type A antitoxin antibody. These experiments have demonstrated that the Japanese quail embryo at 15 days of incubation is an effective vertebrate animal model to detect the activity of botulinum type A toxin. Based on this data the LD50 for quail embryos after 1 day is approximately 1 ug of toxin per kg of body weight and after 3 days is approximately 500 ng of toxin per kg of body weight. Utilization of a quail embryo may be a beneficial model for the analysis of botulinum toxin activity, while decreasing assay assessment time, and enhancing personnel safety.