Abstracts From Fall 1999
1. TITLE: "SERENDIPITY PURIFICATION OF PSILOCYN"
AUTHOR: Arnold Melnikoff, Washington State Patrol Crime Laboratory, Spokane, Washington
ABSTRACT: A chance observation resulted in the conclusion that an impure extract of mushrooms containing psilocyn while being analyzed on a FTIR spectrometer underwent a spontaneous purification. This spontaneous purification was shown to be reproducible in several subsequent extracts of mushrooms from different cases.
This paper will present FTIR spectra of the spontaneous
purification of psilocyn extracts as well as a discussion of the research
which was conducted to try to explain this phenomena.
No arson papers were presented.
1. TITLE: "HOW VETERINARIANS CAN AID WILDLIFE FORENSIC INVESTIGATIONS"
AUTHORS: Walter Cook, Wyoming Game and Fish Department and Todd Cornish, Wyoming State Veterinary Laboratory
ABSTRACT: Veterinarians can offer a great deal of assistance to wildlife forensic investigators. Radiographs can help identify bullets and/or bullet fragments and serve as a permanent record. Their knowledge of anatomy often enables veterinarians to retrieve bullets and fragments with comparatively little damage to the carcass. Occasionally, suspects will claim that they put a sick or injured animal out of its misery. A thorough necropsy can help determine an animal's actual health status prior to death. Such necropsies can also identify the ultimate cause of death. A veterinarian can usually determine which wound was the lethal wound and may be able to determine the temporal order of injuries. It may be possible to determine how old a non-lethal wound is. Veterinary toxicological analysis can often identify a poison used. Necropsies can also identify the species involved in predatory losses of livestock. Additionally, it is often possible to determine if predators killed an animal or if it died due to some other cause and was subsequently scavenged.
Necropsies are generally most useful when the
carcases is fresh and intact (not gutted). It is important that the veterinarian
performing the necropsy have experience in forensics, as this is not part
of the standard veterinary curriculum. We recommend that a system for veterinarians
to become involved in forensic cases be developed a priori and that good
working relationships be fostered among forensic investigators and veterinary
2. TITLE: "MICROSATELLITE ANALYSIS OF WHITE-TAILED DEER"
AUTHORS: Jerry L Ruth, Ph.D., U.S. Fish & Wildlife Forensic Laboratory
ABSTRACT: Forensic analysis in wildlife casework frequently requires the accurate determination of the number of individual animals represented by evidence. This includes both minimum number of individuals ('exclusion') as well as matching two or more samples to the same individual animal ('inclusion'), requiring the analysis of multiple variable genetic loci to draw a reasonable conclusion. Recent advances in genetic knowledge coupled with the increasing need to analyze degraded or otherwise compromised samples have led to the use of polymerase chain reaction ('PCR')-based analysis of simple tandem repeats ('STRs'), or microsatellites, as a measure of genetic variability. This test format uses fluorescent PCR primer sets which bind to specific sequences flanking short (100-400 base pair) variable length stretches of DNA consisting of di- or tetra-nucleotide repeats. The PCR-amplified product is analyzed by electrophoresis, and the fragment size variants observed are used to gauge genetic differences. Although analysis of human microsatellite DNA is well understood and widely accepted in forensic applications, similar analysis of most wildlife species has not been done or is poorly developed.
White-tailed deer (Odocoileus virginianus), whose numbers exceed twenty million animals, range over most of the North American continent, and are the most wildly hunted large game animal. In the United States, white-tailed deer occur in 45 states and two island territories. In an effort to gauge genetic variability, twenty-three known primer sequences derived from deer, cow, and caribou were synthesized and screened against a reference population of more than one hundred individual white -tailed deer using low PCR stringency. Ten primer sets with promising results from these initial trials were tested against a second population of deer, and the annealing temperature and magnesium concentration optimized for maximum signal strength. Three different fluorescent labels were used to allow as many as eight primer sets to be multiplexed in a single PCR reaction. The most promising loci included Cervid 1, BM4107, RT7, RT9, T7, and T159S.
To provide a population database, reference tissue samples from known white-tailed deer populations throughout the United States were collected from focused geographical areas such as national wildlife refuges, wildlife management units, state parks, and controlled hunts. To date, samples from more than two thousand individual deer representing 90+ populations in 43 states have been collected. To establish the variability (number of alleles and allele frequency) at each microsatellite locus, the samples are each being analyzed with a minimum of six distinct primer sets. This database is being used to provide the basis for determining the probability of identity for two or more unknown samples in forensic casework.
The current test format allow the isolation of DNA from blood or tissue without organic extraction, followed by PCR amplification, automated gel electrophoresis, and determination of result for as many as 96 samples in as little as two days. Multiplex PCR (4-6 primer sets per reaction) minimizes reagent use and the amount of sample DNA required. The use of a disposable microtiter tray simplifies handling and tracking, minimizing the risk of sample error.
3. TITLE: "ESTABLISHMENT OF MICROSATELLITE DATABASES FOR FORENSIC CASEWORK IN THE NORTHEAST UNITED STATES: Match Probability Calculations and the Implications of Population Structure"
AUTHORS: Irv Kornfield, University of Maine Wildlife Molecular Forensics Lab, Peter Smith, University of Maine Wildlife Molecular Forensics Lab, and Scott Osgood, Maine Warden Service
ABSTRACT: A DNA forensics laboratory was established in 1997 at the University of Maine to analyze evidence in criminal cases involving wildlife. Protocols are in place for identifying species of a variety of vertebrates, determining gender of mammals, and identifying individual deer and moose. A multiplex PCR reaction has been designed to co-amplify three to four microsatellite loci in a single reaction tube. Allele frequencies multiple microsatellite DNA markers have been determined for population samples of moose and white tail deer from Maine, and databases are now available for use in matching cases. Moose samples can be matched with probabilities ranging from 3.8 x 10-3 to 1.5 x 10-10 and deer samples can be matched with probabilities ranging from 2.1 x 10-3 to 5.8 x 10-12 (assuming the most frequent and the least frequent genotypes, respectively). Genotyping of individual samples has been automated using an ABI 377 DNA Sequencer and GenescanÔ and GenotypeÔ software. These innovations will speed additional regional population surveys and facilitate rapid analysis of evidence in criminal cases.
Population genetic analysis of forensic microsatellite
databases has revealed significant population structure in Maine's deer
herd. Western Maine deer are highly significantly differentiated from coastal
eastern Maine deer. The only apparent barrier to gene flow between these
points is the heavily populated Maine coast. This result has implications
for the management of deer harvest in Maine, as well as the calculation
of match probabilities in cases involving deer.
4. TITLE: "A DNA TYPING SYSTEM FOR PRONGHORN ANTELOPE AND ITS USE IN A POACHING INVESTIGATION FROM WYOMING"
AUTHORS: John W. Coffin, Corey S. Davis, University of Alberta, Edmonton, AB; Deedra Hawk, WY Game and Fish Laboratory, Laramie, WY; and Curtis Strobeck, University of Alberta, Edmonton, AB
ABSTRACT: The Wildlife Conservation Genetics and Forensics Laboratory at the University of Alberta has developed suites of microsatellite (STR) loci for a number of big game species. As a result, this laboratory has a large panel of loci available for testing. Loci developed in one species can often be used in other species. This panel of loci, however, did not prove useful in pronghorn. In order to type this species, a new set of loci had to be developed directly from pronghorn. A suite of six variable dinucleotide loci were selected from the original pronghorn clones, after testing for deviations from Hardy-Weinberg equilibrium and the presence of linkage disequilibrium. These loci were then used to type samples for a database and to analyze submitted case samples.
Six field dressed pronghorn carcasses were discovered
in two separate trash pits and reported. The heads had been removed, but
little or no meat taken. Subsequent investigation led to a skull plate
with horns being taken from a suspect. A DNA match was found between one
of the leg samples and the skull plate. The case went to trial and the
suspect was convicted on six counts of wanton destruction of big or trophy
game. He received 30 days in jail, ca $1300 in fines, $12,000 in restitution
and a 36-year hunting license suspension.
5. TITLE: "COMMERCIAL BROADHEAD EFFECT AND QUANTITATIVE ANALYSIS OF BROADHEAD VS FIREARM WOUNDS IN WHITE-TAILED DEER"
AUTHORS: Michael Stockdale, Forensic and Analytical Specialist, Tennessee Wildlife Resources Agency, Wildlife Forensic Laboratory, Department of Chemistry, University of Tennessee at Martin, Martin, TN
ABSTRACT: Trace metal analysis of commercially produced broadheads was conducted to determine the background level of copper and lead contained on the surface of the broadhead following manufacturing. The level of copper and lead from these broadheads was then compared to known copper and lead values from white-tailed deer to determine if inserting a broadhead into the wound tract would influence the quantitative analysis of the wound tract. Although inserting a broadhead into a firearm wound orifice post-mortem changes the morphological appearance of the wound orifice, it does not influence the analysis of the wound tract when analyzed by flame atomic absorption spectrophotometry. Firearm wounds can be differentiated from arrow wounds quantitatively, using flame atomic absorption spectrophotometry, without concern of broadhead interference affecting the results.
1998 Proc. Annu. Conf. Southeast. Assoc. Fish
and Wildl. Agencies 52:000-000
6. TITLE: "PRELIMINARY REPORT ON IDENTIFICATION OF THE HARVEST SITE OF FRESHWATER MUSSEL SHELLS IN THE UNITED STATES"
AUTHORS: Michael Stockdale, Forensic and Analytical Specialist, Tennessee Wildlife Resources Agency, Wildlife Forensic Laboratory, Department of Chemistry, University of Tennessee; Anthony L. Harmon, Ph.D., Professor, Department of Chemistry, University of Tennessee; Jennifer Jenkins, Teaching Assistant, Department of Chemistry, University of Tennessee; Cindy H. White, Graduate Student, Department of Chemistry, University of Tennessee; Gabriel E. Craig, Graduate Student, Department of Chemistry, Southwest Missouri State University; and Richard H. Black III, Graduate Student, Department of Chemistry, Southwest Missouri State University
ABSTRACT: Nearly 300 species of freshwater mussels are found in the United States. The Mississippi, Ohio, and Tennessee rivers and their major tributaries, especially in the Southeast, exhibit the greatest variety of species. The greatest diversity and abundance of these species are found in Tennessee. Freshwater mussel shells from the U.S. are the primary source of beads for cultured pearl implanting in Japan and China, and they generate up to $60 + million in U.S. sales with a return of $5 billion annually in foreign trade of pearls, fine jewelry and other products.
Washboard (Megalonaias nervosa) and Threeridge
(Amblema plicata) mussel shells are considered primary bead shells for
cultured pearl production. Their harvest is regulated as to collection
sites, size and species. For this study, shells were collected from 31
sites in 9 states. Quantitative trace metal analysis of the shells, using
atomic absorption spectrophotometry, for 10 trace metals was performed
on 3 sections of each shell. Preliminary results indicate a strong correlation
between the harvest site and the trace metal profile of the shell.
7. TITLE: "A FORENSIC TOOL: USING FATTY ACID PROFILE ANALYSIS TO DISTINGUISH WILD FROM FARM-RAISED FISH"
AUTHORS: Beverly A. Villarreal, Law Enforcement Forensic Specialist, Texas Parks and Wildlife Department
ABSTRACT: Effective 30 December 1990, the sale
of wild-caught red drum. Sciaenops ocellatus was banned in the state of
Texas. Practical enforcement of this legislation required a method that
could unambiguously discriminate between wild and farm-raised fish. A fatty
acid profile database, established by analyzing muscle samples of wild
red drum from four major Texas bay systems and of farm-raised re drum from
two aquaculture ventures, indicated that levels of linoleic acid (18:2n-6)
and arachidonic acid (20:4n-6) distinguished wild from farm-raised red
drum. Since 1991, casework has included undercover operations where poaching
rings were infiltrated for a year prior to busting the ring. Results of
sample analyses were used to prosecute the cases in court. Both these cases
involved freshwater fish species, not red drum. Because of the underlying
principles of why fatty acid profile analysis works to distinguish between
wild and farm-raised fish (namely, "you are what you eat"), this forensic
tool is easily extended to other fish species.
8. TITLE: "WHEN PETS GO BAD: A CASE HISTORY OF A FATAL WOLF/DOG HYBRID ON A HUMAN"
AUTHORS: Robert B. Davies, Colorado Division of Wildlife
ABSTRACT: Many people own wolf/dog hybrids as
pets. These animals are involved in bites and attacks of humans. In December
1996, a human fatality occurred as a result of an attack by 2 wolf/dog
hybrids. The animals had a history of aggressive behavior and the male
had been neutered in an attempt to modify his behavior. On the day of the
attack, the wolf/dog hybrids had escaped their pen and had been observed
chasing livestock in the area. The victim died as a result of shock and
massive hemorrhage as a result of the attack. The resulting wounds were
similar of wolf depredation on animals. Characteristics of bear and mountain
lion attacks on humans closely reflect those of prey species.
9. TITLE: "WILDLIFE FORENSICS - A FIELD PERSPECTIVE"
AUTHORS: Dal Schaefer, Colorado Division of Wildlife
ABSTRACT: The value of wildlife forensics and
its various uses will be discussed from the field officer's perspective.
Both laboratory and field techniques will be discussed and examples given
how field officers can better utilize the wildlife forensics capabilities
that are currently available.
10. TITLE: "IDENTIFICATION OF WILDLIFE ANIMAL HAIR FOR FORENSIC CASES"
AUTHORS: Tom D. Moore, Wyoming Game and Fish Laboratory
ABSTRACT: Animal fibers collected from various
sources during wildlife enforcement investigations are frequently submitted
to the Wyoming Game and Fish Laboratory for examination and identification.
Hairs are examined using light microscopy procedures. Characteristics such
as color, shape, length, diameter, medulla and scale type are used to identify
hairs to order, family, genus or perhaps even species. Proteins in the
root ends and sometimes on the shaft have been used in DNA-polymerase chain
reaction techniques to confirm species of origin and gender. A brief review
of procedures and characteristics as well as case examples will be presented.
Species identification of major ungulates - deer, elk, moose, and pronghorn
antelope; trophy game - mountain lion, grizzly and black bear; furbearers
- bobcat, lynx, badger and beaver; and predators - coyote, fox and wolf
will be emphasized.
11. TITLE: "ESTIMATING BONELESS MEAT YIELD OF BIG GAME FOR USE IN FORENSIC INVESTIGATIONS"
AUTHORS: Warrie J. Means, Professor, University of WY, Meat Science; & Tom D. Moore, Supervisor Wyoming Game and Fish Laboratory
ABSTRACT: When one encounters game animals presented
in the processed form (ie. cut and wrapped boneless meat) it is difficult
to determine the actual number of carcasses in possession. The specie and
sex of processed meat may be determined by biochemical means. However,
the actual number of carcasses can only be determined by anatomical identification
of specific muscles and comparison of processed game meat weight to expected
values (presented here).
12. TITLE: "SPECIES IDENTIFICATION OF NORTH AMERICAN WILDLIFE USING PCR-RFLP OF CYTOCHROME B"
AUTHORS: Richard Bischof and Allen Szalanski, Genetic And Forensic Analysis Laboratory, Nebraska Game and Park Commission
ABSTRACT: Mammal and avian DNA samples from various
North American species were obtained from blood, tissue, hair and feathers.
Polymerase Chain Reaction (PCR) was used to amplify a region of the mitochondrial
DNA cytochrome B gene. DNA Sequences of cytochrome B amplicons revealed
sufficient genetic polymorphism for discrimination of most species examined.
PCR- restriction fragment length polymorphism (PCR-RFLP) was conducted
in conjunction with polyacrylamide gel electrophoresis (PAGE) resulting
in species specific banding patterns. Once samples have been analyzed over
the geographical range of each species, we believe that this technique
will provide a robust, efficient and economical species identification
for wildlife forensic purposes.
13. TITLE: "PHYSICAL TECHNIQUES USED IN THE FIELD OR LABORATORY FOR FORENSIC PURPOSES"
AUTHORS: Dave Oates, Nebraska Game & Parks Commission
ABSTRACT: Identification of a wildlife species for most wildlife personnel is not difficult if the "whole" body is present. This may seem impossible when the head, skin, or meat is missing from a carcass.
When a point system existed in the US for waterfowl species, identification of a carcass of certain species was considered important. By use of breast bones left in the carcass, identification to the family is not difficult. Waterfowl normally can't be sexed from breast bones, but this is not true for pheasants and turkeys. Both of these birds can easily be sexed. Domestic or wild turkeys can be identified by breast bone evaluation.
Freshwater fish can be identified from their fillets - by use of color, shape, bones, scales, and myomers to identify to the family, species, length, age, and etc. This is much easier if the fillet has not been cut into two inch sections, a whole fillet works the best.
Time of death seems to be very important especially
when hunting season and hunting hours are "set" for certain species. Some
individuals, prefer hunting at "night", or a day or two earlier than the
opening day. Utilizing temperature, rigor, electrical stimulus, eyes, potassium
levels, a good approximation of the time of death can be obtained. Tools,
techniques, "hands on", explanations, discussions, and etc for time of
death, fillet, and bone identification will be covered.
14. TITLE: "IDENTIFICATION AND QUANTIFICATION OF GAME TISSUE IN PROCESSED MEAT PRODUCTS"
AUTHORS: Paul J. Wilson & Bradley N. White, Wildlife Forensic DNA Laboratory, Trent University, Peterborough, Ontario, K9J 7B8
ABSTRACT: A number of molecular techniques
have been developed to identify the species of origin of unknown tissue
samples in wildlife forensic cases. Illegal commercialization investigations
may require the detection of game meat that has been mixed with domestic
meats, specifically processed meat samples. We have modified an existing
protocol using highly repetitive satellite DNA markers to identify and
subsequently quantify the amount of game tissue present in a processed
meat sample. P32 radioactively labelled genomic DNA of the game species
being detected is hybridized to Southern blotted DNA digested with species
diagnostic restriction enzymes and, following highly stringent washing
conditions, an autoradiograph reveals species-specific banding patterns
with essentially no cross-homology to other game or domestic species. Comparing
signal intensities of satellite DNA bands between case and control samples
of known concentration allows a relative quantification of the game tissue
present in a processed meat sample. Data are presented from an Ontario
Ministry of Natural Resources case that demonstrates the effectiveness
of this technique.