First European interlaboratory comparison of tetracycline and age determination with red fox teeth following oral rabies vaccination programs.

The first European interlaboratory comparison of tetracycline and age determination with red fox (Vulpes vulpes) tooth samples was organized by the European Union Reference Laboratory for rabies. Performance and procedures implemented by member states were compared. These techniques are widely used to monitor bait uptake in European oral rabies vaccination campaigns. A panel of five red fox half-mandibles comprising one weak positive juvenile sample, two positive adult samples, one negative juvenile sample, and one negative adult sample were sent, along with a technical questionnaire, to 12 laboratories participating on a voluntary basis. The results of only three laboratories (25%) were 100% correct. False-negative results were more frequently seen in weak positive juvenile samples (58%) but were infrequent in positive adult samples (4%), probably due to differences in the ease of reading the two groups of teeth. Four laboratories (44%) had correct results for age determination on all samples. Ages were incorrectly identified in both adult and juvenile samples, with 11 and 17% of discordant results, respectively. Analysis of the technical questionnaires in parallel with test results suggested that all laboratories cutting mandible sections between the canine and first premolar obtained false results. All the laboratories using longitudinal rather than transverse sections and those not using a mounting medium also produced false results. Section thickness appeared to affect the results; no mistakes were found in laboratories using sections <150 μm thick. Factors having a potential impact on the success of laboratories were discussed, and recommendations proposed. Such interlaboratory trials underline the importance of using standardized procedures for biomarker detection in oral rabies vaccination campaigns. Several changes can be made to improve analysis quality and increase the comparability of bait uptake frequencies among member states.