-191- David W. Fong Department of Tourism Wildlife Division Government of Newfoundland and Labrador. and

Transcription

1 -191- IDENTIFICATIO~ OF MOOSE MEAT BY IMMUNODIFFUSION David W. Fong Department of Tourism Wildlife Division Government of Newfoundland and Labrador and Bodil Larsen faculty of Medicine Memorial University of Newfoundland Abstract: Moose (Alces alces) specific anti-serum, for meat identification, was developed by injecting a caribou (Rangifer tarandus) with moose albumin and blood serum. In double diffusion tests with blood serum or meat extracts of caribou, deer (Odocoileus virginianus), domestic cow, horse, sheep, pig, goat, guinea pig (Cava cobaya), and rabbit (Oryclolagus cuniculus) no cross reactivity was observed. Several methods have been developed to differentiate members of the deer family through analysis of meat samples for wildlife enforcement purposes. Jackson (1962) used as-' cending paper chromatography patterns of meat extracts to identify members of the deer family. Dilworth and McKenzie (1970) who state that Jackson's methods are not reliable in Atlantic Canada, have used starch-gel electrophoresis patterns of protein, esterase and lactic dehydrogenase from meat extracts to show differences between moose, deer, and caribou. The Royal Canadian Mounted Police Crime Detection Laboratories,

2 -192- which test meat for many Wildlife Enforcement Agencies. have not accepted these methods as reliable (G. Heuchert. Personal Communication). These laboratories use the precipitin reaction to identify meat of the deer family. However. the commercial anti-serum used is not genera spec' fico the taxonomic level of identificatton required for prosecution under certain conditions. Bunch et al (1976) has used analytical polyacrylamide gel isoelectric focusing to show species specific hemoglobin banding patterns of moose. deer. and elk (Cervus canadensis). The purpose of this study was to develop a moose specific anti-serum that could be used to identify moose meat. MATERIALS AND METHODS During this study. animals were located and immobilized (three times) using Cap-Chur darting equipment (Palmer Chemical and Equipment Co. Douglasville. Georgia) from a Bell Jet Ranger Helicopter. Immobilization and revival were brought about using M99 and M50-50 (D-M Pharmaceuticals Rockville Md.) respectively at a dosage of 1 ml per 45 kg of body weight. At the time of the first immobilization. the caribou was fitted with a radio collar to aid in subsequent locatings. Approximately 200 ml of fresh blood was collected from the jugular vein of a moose and transported to the laboratory where the serum was separated by centrifuging at 1000 g for 10 minutes. To isolate moose albumin. ammonium sulfate (0.312 gm/ml)

3 -193- was added to 25 ml of serum, stirred and allowed to equilibrate at 4C for 10 minutes, then centrifuged for 10 minutes at 2000 g. The supernatant was dialized at 4C against two changes of two litres of distilled H 2 0 for 48 hours. dialyzate was subjected to a series of cold Ethar.ol precipitations following the method of Cohen et al (1947). The albumin precipitate was taken up in 25 ml H 2 0, lyophilized, and stored at -10C. The of distilled For immunization, 20 mg of albumin was suspended in 2 ml of 0.9 percent sodium chloride and 2 ml of Freund Complete Adjuant (Bacto, Difco Laboratories, Detroit, Michigan) by vigorous mixing and injecting intramusculary into the inside upper thighs of a caribou. After a three week incubation period a booster injection was given. At this time, 20 mg of albumin was mixed with 2 ml of moose serum and 2 ml of Freund Complete Adjuant. After a further one week of incubation, 50 ml of blood was taken from the caribou and the serum obtained was stored at -10 C until tested. Caribou serum, straight and diluted to 1:8 in saline, was tested for the presence of anitbodies to moose serum proteins by double diffusion in agar (Noble, Difco Laboratories) using moose serum, fresh and frozen meat drippings, obtained by allowing frozen meat to un thaw at room temperature and collecting the runoff, and saline extracts of meat as antigens. Saline extracts were obtained by blend;ng C.2 volumes of 0.9 percent sodium chloride per volume of meat, then cenirifuging at at room temperature. The caribou serum, undiluted and diluted to 1:2, was checked for cross reactivity with

4 -194- caribou. goat, horse, sheep, guinea pig, and rabbit sera, (undiluted and diluted to 1:8). as well as saline extracts and drippings. (both obtained as before). of frozen caribou. beef. pork, and sheep. A blind test for identification of moose meat was conducted on samples of frozen meat from caribou. moose. beef and deer. A total of 26-3mples were tested. All tests were conducted on agar plates with 15/ul wells. the center of the wells having a distance of O.B cm from each other using known moose serum as a control. Approximately 4 ml of 0.2 percent agar in phosphate Buffer Ph 8.0 was pipeted into petri dishes (6 cm X 1 cm) and stored at 1 0 C until used. Wells were made using a small hollow tube having an outside diameter of 4 mm. RESULTS The caribou serum was found to contain antibodies specific to moose serum proteins which could identify moose serum (undiluted and diluted to 1:B). saline extracts. (undiluted and diluted to I:B). and drippings from fresh and frozen moose meat. No positive cross reactivity was found with sera and meat samples of other species tested. In the blind test. 16 samples of moose meat from different moose were identified from the 26 samples tested with no errors or ommissions. DISCUSSION Newfoundland has only two members of the deer family: moose and caribou: the moose specific anti-serum provides a

5 -195- method of differentiating their meat or blood samples for enforcement purposes. The double diffusion test can be set up in less than five minutes using a minimum of equipment and experience. The results can be read at a glance hours later. We feel that this method is a reliable, simple, fast, and inexpensive way to identify moose meat or blood. ACKIIOWL EDGI'IENTS The authors wish to thank R. Maloney of the Biochemistry Department, Memorial University of Newfoundland for isolating the albumin. Also acknowledgment is given to B. Porter of the Wildlife Division, Department of Tourism, Government of Newfoundland for his participation and guidance in the radio telemetry and immobilization of the animals. LITERATURE CITED Bunch, T.D., R.W. Meadows, W.C. Foote, L.N. Egbert and J.J. Spilleti Identification of Ungulate Hemoglobins for Law Enforcement. J. Wildl. Manage. 40(3}: Cohen, E.J., W.L. Huges Jr. and J.H. \ /eare Prepareticn and properties of serum and plasma proteins XIII. Crystallization of Serum Albumins from Ethanol Water Mixtures. JACS. (69) Dilworth, ToG., and J.A. McKenzie Attempts to Identify Meat of Game Animals by Starch-gel Electrophoresis. J. Wildl. Manage. 34(4}:

6 -196- Jackson. C.F Use of Paper Chromatography in Identifying Meat of Game Animals. Management and Research Division. New Hampshire Fish and Game Dept. Tech. Circ pp.