Matson's Laboratory LLC
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Transverse section of raccoon first premolar. The thin section was cut from the intact tooth, without prior treatment, and mounted on a microscope slide. Microscopic examination with illumination by ultraviolet light reveals two fluorescing layers induced by tetracycline administration.
Tetracycline is administered to the animal either by injection or in treated food. Action of the drug results in the formation of an altered tissue layer in tooth and bone. This "biomarker" can be seen in thin, calcified tooth and/or bone sections viewed with a fluorescent microscope.
The presence of biomarker is determined by studying the laboratory preparation, a thin section of calcified tooth or bone. The sectioning is done with a specially equipped saw, and the sections are permanently mounted on a microscope slide.
ESTIMATING POPULATION SIZE. The technique has been used as a "mark-recapture" tool to estimate black bear population size (Garshelis, D. L., and L. G. Visser. 1997. Enumerating megapopulations of wild bears with an ingested biomarker. J. Wildl. Manage. 61:466-480). Conditions necessary for successful application of the technique are discussed in the paper: "The data from Michigan demonstrate that for megapopulations of 5,000 or more animals and annual harvests near 15%, about 200 marks (roughly 600 baits) are necessary to generate an estimate having 95% confidence intervals within 25% of the estimated population size..."
EVALUATING EFFICACY OF VACCINATION OR CONTRACEPTION. Baits contain tetracycline as well as vaccine or contraceptive. After broad-scale application of baited treatments, efficacy is evaluated by examining teeth and/or bone from animals taken within the treated area.
OTHER POTENTIAL APPLICATIONS include animal identification. For example, if a method of mass administration were feasible for cloven hoofed animals (e.g. dosage via treated feed) then a group could be "marked" to detect DISPERSAL. In animals captured for RESEARCH, the permanent biomarker may be useful as a supplemental or substitute identifier. If recapture takes place during summer and after a long enough interval (approximately a month), multiple biomarkers will be detectable. When the tooth is large enough (bear premolar, raccoon canine) a biomarker can sometimes be dated by determining the cementum age at the time it was formed.
Specimens: Seven hairs from Maine raccoons that had tested positive for tetracycline biomarker, and one hair that tested negative, were analyzed to detect the presence of tetracycline fluorescence.
Laboratory methods: Hairs from 8 raccoons were whole mounted in glycerine jelly on microscope slides. Care was taken to include the hair root bulb with each mount. The slide mounting method used was the same used by Matson's for tooth sections to be examined for tetracycline biomarker. Whole mounts were examined under the fluorescent microscope, using the standard tooth section analysis procedure.
Results: No part of any hair in any of the 8 specimens showed tetracycline fluorescence.
Discussion: Tooth sections from 7 of the animals had recent biomarkers (formed within the past 4-8 weeks). If tetracycline or any fluorescing by product was incorporated at all into the hair, fluorescence should have been observed because hair elements present in the specimens were being formed at the time of tetracycline ingestion.
Conclusion: Tetracycline ingestion does not produce a visible fluorescent biomarker in hair.
The calcified tissue reaction with tetracycline produces a clearly visible mark only during the season of most rapid growth. In late fall, winter, and early spring cementum growth is much reduced and there may be no induction of visible biomarker.
The chief reason that attempts fail with biomarkers is inadequate dosage. Evidence suggests that a dosage of 20mg/Kg is ample to induce a clearly visible mark. Adequate dosage has been achieved in studies of black bears by embedding tetracycline capsules inside a 2-lb chunk of bacon. However, attempts to administer adequate tetracycline to ungulates by treating pelleted food have not been successful because the treatment appears to render the food unpalatable. Syringe injections may be effective, but the injected volume is large. Attempts to inject tetracycline by a dart-gun syringe have not been successful. The efficacy of multiple, simultaneous dart-gun injections may deserve consideration and study.
In black bears, the old animal may not reveal a biomarker even if it received an adequate dosage. The rate of cementum production in the old animal is greatly slowed, and may not be great enough to incorporate the amount of reactive material necessary for a clearly visible mark.
Biomarker is also induced in bone but, unlike tooth, remodeling may result in complete loss of the biomarker in 2-3 years.
Administer an ADEQUATE DOSAGE of tetracycline during SUMMER. The PM1 tooth in bears is an acceptable specimen for biomarker screening, but the third incisor (I3) and canine are larger and may have greater biomarker visibility. To compensate for the potentially inadequate biomarker induction in old bears, collect a segment of rib bone along with the tooth. The bone may contain a more clearly visible biomarker until 2-3 years after induction when it is lost because of normal bone remodeling processes.