A simplified protocol for detecting two systemic bait markers (Rhodamine B and iophenoxic acid) in small mammals

Abstract

We developed a method of quantifying levels of fluorescence in the whiskers of wild stoats (Mustela erminea) using fluorescence microscopy and Axiovision 3.0.6.1 software. The method allows for discrimination between natural fluorescence present in or on a whisker, and the fluorescence resulting from the ingestion of the systemic marker Rhodamine B (RB), although some visual judgement is still required. We also developed a new high performance liquid chromatography (HPLC) protocol for detecting the systemic marker iophenoxic acid (IPA) in the blood of laboratory rats (Rattus norvegicus) and wild stoats. With this method, the blood of an animal that has consumed IPA can be tested for the presence of the foreign IPA compound itself. This is a more reliable test than the previous method, which measured the raised level of natural blood protein-bound iodine correlated with IPA absorption. The quantity of blood required from animal subjects is very small (10 μl), so the testing is less intrusive and the method can be extended to smaller species. The extraction technique uses methanol, rather than acids and heavy metal salts, thereby simplifying the procedure. Recovery of IPA is quantitative, giving a highly reliable reading. In experiments on captive rats the IPA method proved successful. Of 12 positively marked carcasses, two that had not been frozen for the 24 h before blood samples were taken showed relatively lower IPA levels. The same IPA detection method, as well as the whisker analysis for RB, was applied successfully to a population of wild stoats to which both Rhodamine B and IPA were made available at bait stations. The presence of both bait markers was detectable in rats for at least 21 days and in stoats for at least 27 days.