2004 Fall Meeting Abstracts

NWAFS 2004 FALL MEETING PRESENTATIONS

ORAL PRESENTATION ABSTRACTS:

Age at Death Estimation Using Human Bone Microstructure
BARNUM, Kori
Department of Anthropology, Portland State University, Portland, Oregon USA

Abstract: The ability to accurately estimate age at death from human skeletal remains is critical to forensic and archaeological analysis; when incomplete or fragmentary remains are encountered, age estimation becomes problematic. Over the last 50 years, several researchers have developed histological techniques for age estimation by quantifying microstructural variables in human long bones such as the femur, tibia, and rib. These techniques require only a cross-section of the long bone shaft rather than the entire element, and rely on age-related variation exhibited by osteons, the basic structural unit of compact bone. The physiological basis for histomorphometric age estimation techniques rests on the distinction between bone modeling and bone remodeling; bone modeling is primarily an activity of development, and the level of bone modeling dramatically decreases after skeletal maturity is reached, while remodeling continues throughout adulthood as an ongoing process of skeletal maintenance. The current research examines the drifting osteon, a microstructure associated with bone modeling and development, in order to assess its utility in histomorphometric age-at-death estimation. Using the right and left clavicles from 30 individuals, the number of drifting osteons encountered, as well as the size of each drifting osteon, was analyzed to determine if age-related trends were demonstrated.

 

Using Geographic Information Systems (GIS) as a Forensic Tool to Predict and Determine the Source of Fishery-Related Marine Mammal Mortality Events
BURDETT, Leslie, and Wayne MCFEE
Center for Coastal Environmental Health and Biomolecular Research (CCEHBR), National Ocean Service (NOS), National Oceanic and Atmospheric Administration (NOAA), 219 Ft. Johnson Rd., Charleston, South Carolina 29412 USA

Abstract: Recently, GIS techniques have been employed to identify fishing gear potentially responsible for marine mammal mortality events. Signature maps of ropes commonly used in South Carolina commercial fisheries were developed using Adobe Photoshop® and ArcGIS®. Rope samples of varied braiding styles were dipped in paint and pressed upon a canvas board to obtain a unique rope marking, or ‘rope signature.’ Digital images of the rope signatures were imported into Photoshop® and converted to black and white. The outline of each converted image was digitized using ArcGIS® to create rope signature maps. These maps were superimposed onto marine mammal stranding photographs to match the signatures to an entanglement wound, similar to fingerprint matching. Additionally, digitized maps were created from entanglement impressions on the epidermis of marine mammals. Photographs of wound impressions on bottlenose dolphins (Tursiops truncatus) and a pygmy sperm whale (Kogia breviceps) were manipulated in Adobe Photoshop® to adjust brightness, color, and contrast. The outlining edge of the wound image was digitized in ArcGIS®, and the resulting impression maps were compared to prototype rope maps to identify the wound’s source. Because fishing gear is often fishery specific, this forensic technique may help predict and mitigate sources of future human interaction. (Keywords: GIS, marine mammals, entanglement, management)

 

Getting DNA from a Rock: A Fishy Tale from the Columbia River
BURNHAM-CURTIS, Mary K.
National Fish and Wildlife Forensics Laboratory, 1490 E. Main St., Ashland, Oregon 97520-1310 USA

Abstract: The white sturgeon, Acipenser transmontanus, is the largest freshwater fish in North America. The market for white sturgeon caviar has grown substantially in scope and value with the collapse of the Russian sturgeon fishery. Poaching of North American sturgeon and paddlefish has brought charges nationwide in the last three years against individuals and companies accused of trafficking in illegally-obtained caviar, violating endangered species laws, and misrepresenting the domestic product as rare Russian caviar. Sport and commercial take of white sturgeon is heavily regulated in all west coast states. In Spring 2002, LE investigators observed an individual landing a large sturgeon below the Dalles Dam during the closed season. The individual was seen dragging the sturgeon across some rocks on the river bank, then loading the fish into a vehicle. Wet rocks were collected from the drag site and sent to the USFWS-NFWFL to determine if sturgeon DNA was present on the rocks. Investigators surmised that since this fish was dragged, some skin cells likely sloughed off and adhered to the slime deposited on the rocks. DNA was successfully extracted from the material on the rocks and sturgeon-specific primers were used to amplify the cytochrome b region of mitochondrial DNA. Sequencing analysis determined that the DNA did indeed originate from white sturgeon. (Keywords: White sturgeon, DNA, caviar, species identification)

 

PCR-Based Protocols for Wildlife DNA Forensics to Determine Species, Gender, and DNA Fingerprinting Profiles
CLARK, AnnMarie, and William G. FARMERIE
ICBR Genetic Analysis Laboratory, University of Florida, 421 Carr Hall, Gainesville, Florida 32611 USA

Abstract: Performing DNA forensics tests for wildlife is often more complicated than it is for humans. In human forensics there is generally no need to determine first if the specimen is human, before proceeding with more detailed identification. Furthermore, in human forensics, there exists a well-developed suite of protocols to determine individual identification and gender. This is not the case for wildlife forensics. Often we must first determine species identity before gender and individual DNA profiles can be completed. At the request of the Florida Fish and Wildlife Conservation Commission, the ICBR Genetic Analysis Laboratory, University of Florida, developed a series of protocols using PCR and restriction enzyme digestion, to identify white tailed deer, Odocoileus virginianus, alligator, Alligator mississippiensis, and wild hog, Sus scrofa. PCR amplification and agarose gel electrophoresis are also used to determine the gender of white tailed deer or turkey samples. Our laboratory is continually expanding our DNA profile database using microsatellite loci for white tailed deer, turkeys, alligators, and hogs. These genetic tests provide wildlife law enforcement officers with powerful tools for dealing with poachers. (Keywords: PCR tests, databases, Whitetailed deer, alligator)

 

Microsatellite Marker Panel for Puma (Puma concolor): Development and Applications for Forensics and Ecology
COLLINS a, c, Julia A., Jay A. WELLa, and Holly B. ERNEST a, b, c
aWildlife and Ecology Unit, Veterinary Genetics Laboratory, University of California, One Shields Avenue, Davis, California 95616 USA
bWildlife Health Center, University of California, One Shields Avenue, Davis, California 95616 USA
cForensic Science Graduate Group, University of California, 1333 Research Park Drive, Davis, California 95616 USA

Abstract: Polymorphic microsatellite DNA markers commonly called Short Tandem Repeats, STRs, in forensic literature) have become important tools of wildlife forensics and molecular ecology by providing accurate means of identifying individuals and characterizing the genetic structure and variation of wildlife populations. Microsatellite markers are particularly useful for studying threatened wildlife species such as puma (Puma concolor, also called mountain lion and cougar), which are highly secretive and dangerous to handle. An accurate method of identification is essential in cases of livestock predation and public safety incidents, as well as cases of poaching and illegal capture. Accurate census and genotype data are needed to properly manage and conserve this protected species. Employing markers originally developed for domestic cats (Felis catus) by the Laboratory of Genetic Diversity, National Cancer Institute, Frederick, MD, we have developed a panel composed of polymorphic microsatellite markers to differentiate individual pumas and provide parentage, kinship, and population structure information. The panel differentiates samples originating from puma from other felids, and provides discriminatory forensic match probability (i.e., likelihood that identical DNA genotypes would be drawn at random a second time from the population). (Keywords: Puma concolor, mountain lion, microsatellite, STR panel)

 

Forensic Identification and Investigation of Hybridization in Marine Mammals using Hemoglobin and Myoglobin Molecular Weight Markers
DUFFIELDa, Deborah A., Edgard O. ESPINOZAb, and Nélio B. BARROSc
aDepartment of Biology, Portland State University, P.O. Box 751, Portland, Oregon 97207 USA
bNational Fish and Wildlife Forensics Laboratory, 1490 E. Main St., Ashland, Oregon 97520-1310 USA
cMote Marine Laboratory, 1600 Ken Thompson Parkway, Sarasota, Florida 34236 USA

Abstract: Hemoglobin and myoglobin molecular weights and electrophoretic variation have been used to distinguish marine mammal species and ecotypes. Using molecular weights of the two molecules in concert, we have been able to differentiate nearly 50 marine mammal species to date. These are incorporated into the National Fish and Wildlife Forensics Laboratory (Ashland, Oregon) database for the diagnostic determination of species falling under the protection of CITES treaties. Hemoglobin and myoglobin molecular weights have also been used to confirm species identification of Kogia breviceps vs. K. sima in stranding situations and hemoglobin electrophoresis to distinguish coastal vs. offshore Tursiops truncatus ecotypes. These techniques have further proved useful in the detection of hybridization involving a number of cetacean and pinniped species. Molecular weight differences have been detected using either ESI MS or MALDI-TOF mass spectrometry. Both mass spectrometry and electrophoretic methods are rapid procedures, robust to molecular degradation (especially mass spectrometry) and require only very small samples which can be stored frozen or dried. We emphasize the application of these biomarkers for identifying species in situations where field identification of individual specimens is doubtful or confusing (i.e., strandings), for forensic determination, and for the detection of interspecies hybridization. (Keywords: molecular weight biomarkers, marine mammals)

 

Identification of Resizing Die Marks on .38 Special and .357 Magnum Cartridge Cases
EVANS, Samantha
California Department of Justice, Santa Rosa Laboratory, 7505 Sonoma Hwy, Santa Rosa, California 95409 USA

Abstract: Reloading is not a hobby that is unique to the United States—approximately 15% of annual sales by RCBS, a U.S. reloading equipment and supply manufacturer, are to buyers in Europe and Australia. Since reloading reaches across communities, cultures, and borders, it is expected that reloaded ammunition will appear as evidence in a case submitted to a forensic laboratory. In this study, thirty brass and nickel .357 Magnum and thirty brass and nickel .38 Special cartridge cases were resized with an RCBS steel resizing die, reloaded, and fired with one of three powder loads (standard, +P, or magnum). The steel RCBS resizing die was found to have a unique surface that left individual marks on the surface of the cartridge case. These marks were not affected significantly by the reloading process or firing, and the resized cartridge cases could be identified back to the steel resizing die. Seven .357 Magnum cartridge cases were also resized with an RCBS carbide resizing die, reloaded, and fired. The carbide die produced very few marks on the cartridge case and the cartridge cases could not be linked back to the die. (Keywords: Reloading, resizing die, cartridge cases)

 

Sex-Specific Patterns of Genetic Variation in North American Gray Wolf Populations: A Comparison of Y-Chromosome Short Tandem Repeats and mtDNA
FAIN, Steven R., and Dyan J. STRAUGHAN
National Fish and Wildlife Forensics Laboratory, 1490 E. Main St., Ashland, Oregon 97520-1310 USA

Abstract: Six Y-linked short-tandem-repeat (STR) loci (MS34A, MS34B, MS41A, MS41B, 990 35.4 and 650 79.2) were analyzed in 300 male gray wolves from five populations in North America. These comprised three subspecies from Alaska, Western Canada and the Eastern, Western and Southwestern Distinct Population Segments (DPS) designated by the USFWS under the Endangered Species Act, and included individuals from both the Northern US Rockies and Mexican wolf experimental reintroduction populations. The results were compared with 237 base pairs of mtDNA control region (CR) sequence data obtained from the same individuals in order to study possible differences in male versus female patterns of genetic variation. Y-STR haplotype diversity was higher than mtDNA genetic diversity in all five populations with at least fourteen Y-STR haplotypes being exclusive to single populations. While four mtDNA CR haplotypes were commonly observed across all North American wolf populations except Southwestern wolves, a fifth haplotype was unique to Western wolves and a sixth distinguished Southwestern wolves from all others. Two mtDNA CR haplotypes, commonly observed and exclusive to Eastern wolves, were most similar to those of coyotes. (Keywords: Gray wolves, Y-STR, mtDNA sequence)

 

Forensic Application of Short Tandem Repeat (STR) Analysis to North American Elk (Cervus elaphus) Populations
HAMLIN, Brian C.
National Fish and Wildlife Forensics Laboratory, 1490 E. Main St., Ashland, Oregon 97520-1310 USA

Abstract: North American Elk (Cervus elaphus) are one of the largest and most sought after game mammals on the continent and one of several cervids routinely submitted to NFWFL for individual identity. As the forensic support group for United States Fish & Wildlife Service Special Agents, among a list of other constituents, we are mandated to provide expertise and support for wildlife law enforcement investigations. This often requires a significant review of the published literature, coordination with individuals and agencies in the field for reference standards collection, and additional research at the lab bench. An evaluation of North American Elk was undertaken to address questions regarding the individual identity of crime scene evidence and custody evidence. Over 32 populations representing 15 states, two Canadian Provinces and more than 1,500 individual free ranging Elk were analyzed using seven STR loci. Population genetics will be discussed in terms of their forensic application, Probability of Identity development, and future research needs. (Keywords: Elk, STRs, population genetics)

 

Front Page Wildlife Poaching Cases from the Wyoming Game and Fish and the Colorado Division of Wildlife
HAWK, Dee Dee, and Scott SHARPE
Wyoming Game and Fish Laboratory, Biosciences Bld., Department 3312, 1000 E. University Ave, Laramie, Wyoming 82071 USA

Abstract: During the last couple of years, the Wyoming Game & Fish Forensic Laboratory has been involved in several high-profile poaching cases in Wyoming and in Colorado. This paper will discuss several of these cases and demonstrate the different testing protocols used to determine the guilt or innocence of numerous suspect poachers. One case to be discussed is the poaching of trophy mule deer from the wrong area and the planting of evidence in a different area to disguise the crime. The laboratory tested samples from two separate bighorn sheep cases from Colorado that resulted in over $58,000 in fines and restitution. MALDI-TOF analysis was used in a case where six different defendants are being investigated/charged with numerous poaching allegations. MALDI was also used to demonstrate a suspect poacher was telling the truth. This year, the Wyoming laboratory brought mitochondrial DNA analysis on-line for species identification. It was just in time to determine if a meat processor was using beef in his “wild game” jerky. (Keywords: Poaching, microsatellites, PCR)

 

Forensic Ornithology at the Smithsonian Institution: Introduction to the Feather Identification Lab
HEACKER, Marcy
Division of Birds, E600, MRC 116, 10th & Constitution Ave, NW, Smithsonian Institution, National Museum of Natural History, Washington D.C. 20560 USA

Abstract: The Feather Identification Lab at the Smithsonian Institution, National Museum of Natural History has provided species identification services for over 30 years. Bird identification methods utilizing whole and microscopic feather characters are used for a variety of applications such as: birdstrikes (bird/aircraft collisions), anthropology artifacts, law enforcement, prey remains, and food contaminants.

Despite the volume of cases and variety of evidence, the approach to species identification is consistent. Whole feathers provide clues to color, pattern and size of a bird; plumulaceous (downy) feather barbules suggest the group/type of bird; and circumstantial information help interpret temporal, environmental and behavioral aspects of a species. Final identifications are based on the combination of these clues along with direct comparison with vouchered museum specimens. In addition to morphological identification techniques, the Feather Lab has begun establishing molecular protocols and methods to enhance current ID capabilities.

While the lab emphasis is service oriented, studies of feather structure are also conducted. Applying statistical and phylogenetic analysis to feather micro-morphology continues to document and examine variation of the plumulaceous feather region to further understand the significance and limits of these structures. (Keywords: Feather, ornithology, morphology, museum, bird)

 

The Cost of Maintaining a Quality Assurance Program
HEGDAHL, Darrell
National Fish and Wildlife Forensics Laboratory, 1490 E. Main St., Ashland, Oregon 97520-1310 USA

Abstract: A formalized and documented quality assurance program supports confidence in the results of forensic analysis and is critical to maintaining an unbiased and credible presentation of fact. Audits, reviews, protocol development/validation, comparative standards preparation/verification, equipment maintenance/calibration, proficiency testing and laboratory accreditation are all components of a quality assurance program which cost time and money. The National Fish and Wildlife Forensics Laboratory has a formalized and documented quality assurance program and has been accredited through the American Society of Crime Lab Directors, Laboratory Accreditation Board since 1997. This presentation will review the cost of maintaining this program.

 

Species Identification of Mammalian Horn by PCR
HOESCH, Robert M., and Steven R. FAIN
National Fish and Wildlife Forensics Laboratory, 1490 E. Main St., Ashland, Oregon 97520-1310 USA

Abstract: In the effort to enforce laws regarding the importation of parts and products from the critically endangered rhinoceros species, we have developed a method for distinguishing between true rhinoceros horns and those from various domestic and wild bovid species. The method relies on extraction of the minimal amount of DNA remaining in horn and amplification and direct sequencing of a portion of the mitochondrial Cytochrome b gene. The sequence is subjected to phylogenetic analysis in comparison with a database of representative rhinoceros and bovid species. The commercial value of true rhino horn is high enough so that the incentive for fraud is significant. This report will discuss recent efforts to develop species specific primers intended to screen out common bovids and avoid the time and expense of direct sequencing. In addition, some bovid species may themselves be endangered in their native ranges, and the identification of these might aid conservation efforts; many of these endangered bovid species have domestic forms as well, which at the molecular level may not be distinguishable. The implications for wildlife law enforcement will be discussed. (Keywords: Rhino, horn, PCR, bovid)

 

Computer Forensics in the U.S. Fish & Wildlife Service
HORNE, Brian
National Fish and Wildlife Forensics Laboratory, 1490 E. Main St., Ashland, Oregon 97520-1310 USA

Abstract: This talk will give an overview of how computer forensics is being implemented in the U.S. Fish & Wildlife Service, to include a historical perspective of the work done to date, a review of current tools in use, and examples of how information is presented back to the agents for review. (Keywords: Computer, ILook, Encase, E-mail Examiner, Net Analysis)

 

Bringing Non-Human Forensic Testing into Compliance: Solutions and Challenges
KETCHUM, M., and P. GAROFANO
DNA Diagnostics dba Shelterwood Laboratories, P.O. Box 455, 626 Bear Drive, Timpson, Texas 75975 USA

Abstract: For some time now, animal and plant DNA has been admitted into court. Challenges have begun though and now, there have been and will be cases appealed due to the fact that quality control and standardization present in most human forensic laboratories have not been present in many laboratories dealing with non-human species. There have been difficulties with the lack of proficiencies, the lack of animal related CE, standardization between laboratories, audits of laboratories, accreditation and a myriad of other difficulties. In general, there is a breakdown of procedural issues that could jeopardize our cases given a knowledgeable attorney. With this in mind, a group of us have established a new organization, the International Academy of Plant and Animal Forensic Science. With this organization, we are addressing all the issues needed to help a laboratory doing non-human testing achieve both IAPAFS and /or ASCLD accreditation. At this time there are about 100 members worldwide. As far as the USA and Canada, all of the members are currently government (i.e., State or Federally employed) with the exception of only two private laboratories. (Keywords: IAPAFS, forensic testing)

 

Combining Novel Techniques to Achieve Consistent Results in Cases Where Degraded Human / Animal Mixes are Present in the Same Sample
KETCHUM, M., and M. KONIEZNY
DNA Diagnostics dba Shelterwood Laboratories, P.O. Box 455, 626 Bear Drive, Timpson, Texas 75975 USA

Abstract: When presented with a case where there are only minute quantities of DNA present and even the small, degraded quantity that is present is further diluted due to a mixture of human and animal DNA, approaches to analyzing the case become extremely tedious. Combining a number of novel or uncommon approaches to amplification can oftentimes yield not only desired results, but could be commonly incorporated in the laboratory to be used on a routine basis to enable analysis of cases that otherwise would not yield consistent and court-worthy results. One such example is the use of nested PCR for mitochondrial analysis. Though extreme caution must be used in order to prevent contamination, when using the proper protective equipment and techniques, it is easy to take a number of small sequence fragments generated by nested PCR, sequence them, and then combine them in order to achieve the entire mitotype. When seeking either human and/or animal DNA in a mixed sample, we chose to use universal mammalian primers, amplify the DNA and then utilized a yield gel to separate the amplified products into various different sized bands according to species. The bands or the regions where the bands should appear were then excised and purified using the Qiagen™ Gel Purification system. The amplified DNA was then reamplified using primers that generated fragment lengths of between 150 and 200 base pairs. Even when bands were barely visible and sometimes when no visible band appeared on the yield gel, reamplification yielded products in most cases that were viable for sequencing and had consistent reproducible results in controls and evidence as well as blank controls. This is but one of several techniques including dehydration of extractions that can be utilized to achieve satisfactory analysis in such cases.

 

Resolving Isomeric Hemoglobin Phenotypes for Species Identification Employing MALDI-TOF Mass Spectrometry
KIRMSa, Mark A., and Lynn M. KIRMSb
aNational Fish and Wildlife Forensics Laboratory, 1490 E. Main St., Ashland, Oregon 97520-1310 USA
bDepartment of Chemistry, 1250 Siskiyou Blvd., Southern Oregon University, Ashland, Oregon 97520 USA

Abstract: Matrix assisted LASER desorption ionization (MALDI) time of flight (TOF) mass spectrometry is an effective analytical tool for the analysis of proteins. We have found that the analysis of hemoglobin in blood and tissue samples by MALDI-TOF mass spectrometry enables differentiation of one species from another in a straight-forward fashion. The molecular weights of the a-globins and b-globins which are present in blood provide a platform for differentiating the hemoglobin phenotypes present within species. In a few instances, the hemoglobins of two different species are isomeric, and, as such, the molecular weights of their a- and b-globins can not be distinguished directly from MADI-TOF mass spectrometry. In these cases, treatment of MALDI samples with hexafluorophosphoric acid (HPF6) generates adducts of the hemoglobin proteins which allows for differentiation of the isomeric a- and b-globins as a result of their differing primary structures. (Keywords: MALDI, species, identification, isomeric, hemoglobin)

 

Forensic Identification of North American Moose (Alces alces) Using Short Tandem Repeats (STR)
LeMAY, James P.
National Fish and Wildlife Forensics Laboratory, 1490 E. Main St., Ashland, Oregon 97520-1310 USA

Abstract: North American Moose are valued for their massive antlers as a trophy and also as a food source. The hunting of Moose is controlled by State, Federal and Tribal regulations. When laws are violated it is often very difficult for the law enforcement officer in the field to tie the victim, crime scene and suspect together. The National Fish and Wildlife Forensics Laboratory is dedicated to assist law enforcement officers by analyzing biological evidence items to assist in the exoneration or conviction of the suspect. The use of genetic analysis is often vital to the investigation and may be the only source of information that will help the officer in the field. Eighteen sets of STR primers were screened and 10 sets were chosen that amplified well, gave a sufficient number of alleles and displayed enough genetic heterogeneity to allow for an adequate probability of identity to be determined. Moose reference samples were obtained from ten states and two Canadian Provinces. Resulting analysis will be discussed. (Keywords: Alces alces, Moose, STR, microsatellite, probability of identity)

 

Improved Molecular and Statistical Approaches in Wildlife Forensics: Applications in Wild Sheep Conservation
LUIKART, Gordon, Eve ZEYL, Celia MAUDET, Stephanie MANEL, and Pierre TABERLET
Laboratory of Population Genomics and Biodiversity, University of Grenoble, Grenoble, Cedex 09, 38041 France

Abstract: Highly polymorphic DNA markers and new statistical methods (e.g., “assignment tests”) can help to detect and thereby reduce poaching. Assignment tests can identify the population of origin of individuals if populations are genetically differentiated. We describe and evaluate the usefulness of assignment tests for forensic applications using large empirical (microsatellite DNA) data sets from 10 species, plus computer simulations. The Bayesian assignment test of Pritchard et al. (2000) performed better than the exclusion test of Cornuet et al. (1999); but it requires the assumption that the true population of origin was sampled. The median percentage of individuals correctly assigned for the ten empirical data sets was 61% for Pritchard’s test. Both the empirical and simulated data sets suggest that nearly all individuals can be assigned with high statistical certainty (99.9%) for two highly differentiated populations (Fst = 0.15-0.2) when using 10 loci (H>0.6) and samples of 30-50 individuals. Both tests should be used when the true population of origin might not have been sampled. Finally, we illustrate the application of non-invasive sampling to establish genotype data bases and standard forensic tests for detecting illegal trafficking of endangered Urial sheep (Ovis orientalis) from Asia, and bighorn sheep from North America. (Keywords: Assignment tests, identity matching, trafficking)

 

An Efficient Multiplex PCR Approach to Identification of Species and Geographic Origin of Body Parts from CITES Listed and / or Legislatively Protected Shark Species
MAGNUSSEN, Jennifer E., and Mahmood S. SHIVJI
Guy Harvey Research Institute, 8000 North Ocean Drive, Nova Southeastern University, Dania Beach, Florida 33004 USA

Abstract: Heavy exploitation of sharks globally to satisfy the demands of the international fin market have resulted in trade in a few species considered particularly sensitive being restricted or controlled by national legislation or international accord (e.g., CITES). Despite these conservation efforts, however, trade in these species continues because law enforcement monitoring and surveillance is hindered by species identification problems. We present the development and application of a highly streamlined, robust, multiplex PCR assay for identification of basking shark (Cetorhinus maximus; CITES Appendix II) and sand tiger shark (Carcharias taurus; protected in the U.S. and Australia) body parts in the trade. Given the spatially “patchy” nature of national protective efforts for some species, identifying the geographic origin of the traded products will be needed for legal enforcement, and will be informative for assessing geographic trends in exploitation pressure. To this end, we will present preliminary data on development of a multiplex PCR assay that simultaneously distinguishes species and ocean-basin of origin for the sand tiger shark. (Keywords: Shark trade, DNA identification, shark fins)

 

Development of STR Loci for Casework and Management of California’s Elk (Cervus elaphus) Populations
MEREDITHa, Erin, Jeff RODZENa, Jim BANKSa, Ken LEVINEa, and Bernie MAYb
aCalifornia Department of Fish and Game, Wildlife Forensics Laboratory, 1701 Nimbus Road, Suite D, Rancho Cordova, California 95670 USA
bDepartment of Animal Science, University of California – Davis, One Shields Avenue, Davis, California 95616 USA

Abstract: California is home to three subspecies of Elk: the Tule, Roosevelt, and Rocky Mountain. The Tule elk have experienced a severe bottleneck and are now intensively managed and highly fragmented across their original range. A set of 21 polymorphic tetranucleotide STR loci has been produced for casework and population management. Six hundred and seventy-six (676) elk from the three subspecies were genotyped using 16 loci. Highly significant differences in allele frequencies were found between the three subspecies and among Tule elk herds. The past genetic bottleneck experienced by the Tule elk has resulted in significantly less genetic variation and heterozygosity excess when compared to Roosevelt and Rocky Mountain elk. Elk from Modoc and Siskiyou counties have an uncertain taxonomic status, and the data showed Modoc County to contain both Rocky Mountain and Roosevelt elk while Siskiyou County contains predominantly Roosevelt elk and possible subspecies hybrids. Assignment testing of elk to the Tule versus non-Tule lineage is 100% accurate when data from all 16 loci are used. The reported loci will also be useful for individual identification in poaching cases as the probability of identity was 4.04 x 10-11 across the three subspecies and 7.64 x 10-7 within the Tule elk. (Keywords: Elk, Cervus elaphus, microsatellite, individual identification, population genetics)

 

Phylogeography, Genetic Diversity and Conservation Genetic Approach in Three Toed Sloths: Bradypus torquatus and Bradypus variegates
MORAES-BARROS a, b, Nadia, Cristina Yumi MIYAKIb, João Stenghel MORGANTE a, b
Laboratório de Biologia Evolutiva e Conservação de Vertebradosa, Departamento de Biologiab, Instituto de Biociências da Universidade de São Paulo, Rua do Matão 277, 05508-900, São Paulo/SP Brazil

Abstract: Bradypus torquatus is endemic to the Atlantic Forest and it is considered endangered due to deforestation. A more widespread sloth, Bradypus variegatus, occurs in both the Atlantic and Amazon Forest. It is not considered threatened. However, its range includes forested areas in Brazil that are being fragmented. Here we present the results obtained in a genetic study of these two species. Analyses performed using nuclear and mitochondrial DNA indicated low levels of genetic variability and great divergence among populations for both species. In these two species, analyses of mitochondrial DNA show that the Atlantic forest is subdivided into a North and South genetic component. The 75 sampled B. variegatus are grouped in seven distinct mitochondrial phylogroups distributed according to the different sampled regions. One of these phylogroups is in the northeastern region of Brazil and contains haplotypes characteristics of Amazon and Atlantic forests. The most divergent group, representative of the region between Tapajós and Amazonas River, shows values of nucleotide distance higher than those found when comparing the two sloth species, suggesting that additional ecological, morphological, and molecular studies are needed to evaluate how many phylogenetic and evolutionary lineages represent the entire genetic diversity of sloth species. (Keywords: Conservation genetics, biogeography)

 

Overview of the Veterinary Medical Examinations Performed at the National Fish and Wildlife Forensics Laboratory (1990-2004)
RALSTON, Rhoda M., and Richard K. STROUD
National Fish and Wildlife Forensics Laboratory, 1490 E. Main St., Ashland, Oregon 97520-1310 USA

Abstract: Since 1990, approximately 6500 veterinary medical examinations have been performed at the National Fish and Wildlife Forensics Laboratory. Veterinary medical examinations are performed on evidence items submitted by both Federal and State law enforcement officers pursuing wildlife criminal investigations. Veterinary medical examinations may involve either gross necropsy examinations (n = 6099) or radiographic (x-ray) examinations of incomplete carcasses or their parts (n = 400). The necropsy examinations (n = 6100) performed on the wildlife carcasses submitted to the laboratory resulted in the following diagnoses: poisoning (30%), gunshot (27%), trauma (12%), electrocution (6%), oil contamination (4%), entrapment (1%), natural causes (6%), and undetermined (14%). Specific criteria for each diagnostic category are presented.

 

Development of STR Loci for Casework Involving Illegal Take of Mule Deer (Odocoileus hemionus) in California
RODZEN, Jeff, Erin MEREDITH, Jim BANKS, and Ken LEVINE
California Department of Fish and Game, Wildlife Forensics Laboratory, 1701 Nimbus Road, Suite D, Rancho Cordova, California 95670 USA

Abstract: Twenty tetranucleotide STR loci have been developed for mule deer and currently a suite of eight loci are used for individual identification with a probability of identity of approximately 6.25 x 10-10 with allele numbers ranging from five to 15. A set of 100 deer have been multiplexed and genotyped on the ABI-310 and MJ Research “Basestation” and duplexed and genotyped on the ABI-377 and Elchrom system to ensure consistency of genotyping across multiple platforms. A study of 659 deer from across California revealed that some loci exhibited moderate heterozygote deficiencies, suggesting population structure exists. Herd boundaries are basically unknown and could not be estimated from the genetic data, though population genetic analyses suggest an isolation-by-distance scenario exists within California.

The CDFG Wildlife Forensic Laboratory successfully passed a Kelly-Frye challenge for the mule deer DNA individualization procedure and it is now accepted in the California Appellate court level. A Standard Operating Procedure for CDFG’s forensic casework requiring deer DNA analysis was created to codify the methodology using the Elchrom system to standardize laboratory procedures in the event of the loss of key personnel proficient in the technique, and provides the judiciary with a standardized procedure often requested in discovery motions. (Keywords: Deer, Odocoileus hemionus, microsatellite, individual identification, population genetics)

 

Techniques for Field Screening of Firearms Evidence
SCANLAN, Michael
National Fish and Wildlife Forensics Laboratory, 1490 E. Main St., Ashland, Oregon 97520-1310 USA

Abstract: Techniques for in-the-field screening of firearms evidence will be presented. Included will be methods of determining the caliber and rifling characteristics of a bullet, and comparing the results to a database of possible cartridges and firearms that the bullet could have been fired from. Screening of rimfire cartridge cases, to determine if the cases could have been discharged in the suspect’s firearm, or if multiple firearms were used, will also be covered. (Keywords: Firearms, bullets, cartridge cases, GRC)

 

Applying Streamlined Genetic Identification of Shark Body Parts in U.S. Fisheries Enforcement and Monitoring Global-Scale Trade
SHIVJI, Mahmood S., Jennifer E. MAGNUSSEN, and Shelley C. CLARKE
Guy Harvey Research Institute, 8000 North Ocean Drive, Nova Southeastern University, Dania Beach, Florida 33004 USA

Abstract: Large declines in commercially fished shark stocks in the face of burgeoning demand for shark products, especially fins, has resulted in international concerns about sustainability and an expanded list of limited and prohibited (from fishing) shark species in the territorial waters of several countries. Additionally, because individual shark species respond differently to exploitation, there is a strong need for management and conservation efforts on a species-specific basis. Monitoring shark catch and trade and assessing population impacts by species is essentially non-existent, however, due to substantial difficulties in identification of landed shark carcasses and detached fins. Although DNA-based forensics for marine wildlife are being developed, the procedures available thus far (mainly RFLP and phylogenetic approaches) are too time-consuming and expensive for routine management, law enforcement and conservation applications. To address this issue, we have developed a highly streamlined, multiplex PCR-based approach that can discriminate between multiple (up to nine) shark species simultaneously with a single-tube PCR. We report on the diagnostic assay, and demonstrate its use in U.S. NOAA fisheries law enforcement, global scale identification of products from CITES and/or IUCN shark species of concern, and characterization of the Hong Kong shark fin trade. (Keywords: Sharks, fins, law enforcement, CITES, DNA)

 

Forensic Identification of the River Sturgeon Scaphirhynchus
STRAUGHAN, Dyan J., and Steven R. FAIN
National Fish and Wildlife Forensics Laboratory, 1490 E. Main St., Ashland, Oregon 97520-1310 USA

Abstract: Scaphirhynchus is an exclusively freshwater sturgeon in the Mississippi and Mobile River drainages. Two of the three species in the genus, the pallid sturgeon (S. albus) and the Alabama sturgeon (S. suttkusi), are listed as endangered under the ESA, while the shovelnose sturgeon (S. platorhynchus) is of commercial importance. As such, it is important to know which species are being traded and the proportion of the trade they comprise. We sought to develop a DNA procedure for the forensic identification of the caviar and meat of Scaphirhynchus. We characterized sequence variation in a portion of both the cytochrome b gene and the mitochondrial control region (CR) in 119 shovelnose, 26 pallid and three Alabama sturgeon. Comparisons revealed a total of five unique sequences for cytochrome b and 38 CR haplotypes. Alabama sturgeon sequences and representative sequences from pallid and shovelnose sturgeon were identical with respect to the cytochrome b sequences, making it difficult to identify shovelnose to the exclusion of pallid and Alabama sturgeon. Our results also support Campton et. al. (2000) that CR haplotypes reflect a close relationship between pallid and shovelnose sturgeon with no diagnostic base-pair substitutions. However, although our results support their findings of a ‘unique’ Alabama sturgeon haplotype, the potential of its use as a diagnostic genetic character is questioned. (Keywords: cytochrome b, control region, Scaphirhynchus)

 

Patterns of Illegal Wildlife Poisoning in the United States – 1990 to 2004
STROUD, Richard K., Rhoda M. RALSTON, Mark A. KIRMS, and Pamela MCCLURE
National Fish and Wildlife Forensics Laboratory, 1490 E. Main St., Ashland, Oregon 97520-1310 USA

Abstract: Birds and mammals were submitted to the National Wildlife Forensics Laboratory for determination of cause of death by the Veterinary Medical Examiners. In suspected poisoning cases, the digestive tract contents were collected for identification of food items and analysis for specific pesticides or other poison residue. Samples of the digestive tract contents were extracted and screened for pesticide residue using various standard analytical techniques. The incidence of carbamate and organophosphate pesticides along with other acute poisons are reported for 391 cases (1475 carcasses examined). Pesticide poisoning accounted for 22% of deaths of 1402 bald and golden eagles examined. Carbofuran was the most frequently encountered pesticide followed by famphur, aldicarb and strychnine. Examples of typical wildlife poisoning scenarios which vary with different pesticides and effected species are presented.

 

Amazonian Feather Art in the Context of Wildlife Law Enforcement
TRAIL, Pepper W.
National Fish and Wildlife Forensics Laboratory, 1490 E. Main St., Ashland, Oregon 97520-1310 USA

Abstract: For countless generations, indigenous South American peoples have used the feathers of rainforest birds to create objects of great cultural significance and aesthetic appeal. This cultural use of wildlife was sustainable as traditionally practiced. In recent years, however, commercial trade in Amazonian feather art has developed, with sales to tourists in South America and to collectors in North America, Europe, and Asia. Many of the birds whose feathers are used in these objects, including Hyacinth Macaws and Harpy Eagles, are in serious decline, and are protected by national laws and international treaties. Nevertheless, illegal international trade in Amazonian feather art continues. I analyzed over 500 Amazonian feather art objects seized in the course of U.S. Fish & Wildlife Service law enforcement investigations. These items contained the feathers of a minimum of 46 bird species, 23 (50%) of which are protected by United States laws and/or the Convention on International Trade in Endangered Species (CITES). By far the most commonly used feathers were those of the strictly protected Scarlet Macaw (Ara macao), found in 69% of objects examined. Almost all items (92%) contained the feathers of at least one protected species. This analysis confirms that virtually all Amazonian feather artifacts include threatened bird species, and suggests that this illegal trade could jeopardize vulnerable wild populations.

 

Microbial Community Profiling and Characterisation (MCPC) as a Tool in Forensic Analysis
VAN HAERINGEN, Hein, and Henk PANNEMAN
Van Haeringen Laboratorium BV, P.O. Box 408, Wageningen, 6700 AK, The Netherlands

Abstract: MCPC is a fast, universal, robust and flexible technique based on T-RFLP to establish detailed microbial profiles from virtually any sample type including soil, (salt) water and faeces. Semi-quantitative microbial population profiles can be determined in less then 24 hours and are visualised as a peak patterns. Using a custom made database the peaks in the pattern can be related to known micro-organisms. MCPC is highly suitable for studies aiming to compare the microbial composition between different samples. Such comparisons have already been successfully used to provide associative evidence in criminal cases. For the visualisation and comparison of MCPC profiles we have developed dedicated software. The software integrates flexible data visualisation and transformation, automatic grouping and sorting of samples and various heuristic and advanced multivariate statistical methods to analyse complex profiles. Furthermore, data and analysis results can be exported from the programme as delimited text format to facilitate the analysis of the data using other programmes. MCPC can also be useful in cases where there is no clue as to what micro-organisms are present in a sample. A quick and reliable indication of the strain(s) present in a specific sample can be provided, even when the micro-organisms in the sample are hard to culture. MCPC is protected by patents and patent applications. (Keywords: Microbial profiling, T-RFLP, microbial typing, profile comparison)

 

Wildlife Genetics Proficiency Testing Program
VOIN, Doina
National Fish and Wildlife Forensics Laboratory, 1490 E. Main St., Ashland, Oregon 97520-1310 USA

Abstract: Proficiency testing is an integral part of an effective quality assurance program, and is a reliable method of verifying that the laboratory’s technical procedures are valid and the quality of each examiner’s work is being maintained. It is essential that proficiency tests be properly designed, appropriately administered, and fairly evaluated. The Wildlife Proficiency Testing Program (formerly known as Round Robin Wildlife Program) was established to promote quality assurance among wildlife forensics laboratories and to have a common forum for scientific discussion. The intent of the Wildlife Proficiency Program is to ensure that the Genetic proficiency tests reflect the multi-species challenges that are encountered in wildlife forensic science.

 

DNA-Based Wildlife Forensics at the Washington Department of Fish and Wildlife: A General Introduction and Brief Discussion of Elk Populations
WARHEIT, Kenneth I.
Washington Department of Fish and Wildlife, Wildlife Research Division, 600 Capitol Way N., Olympia, Washington 98501-1091 USA

Abstract: The Washington Department of Fish and Wildlife has conducted DNA-based genetics research since 1997. The bulk of this work has concerned salmonid stock identification, but there has been also a substantive effort related to wildlife conservation and population concerns. Common murre, western-gray squirrel, fisher, cougar, and bear are some of the wildlife taxa we have worked with, although most of our efforts have focused on deer, elk, sharp-tailed grouse, and pygmy rabbit. We have over 6000 wildlife samples accessioned into our collection, representing 18 species; over 2500 of these samples are from black-tailed/mule deer, and 1075 from elk. We have worked with a variety of sample material, from fresh liver to hair, scat, bloodstain, and museum skins. Also, as a service to our fish and wildlife law enforcement staff, we have recently begun wildlife-related forensics work; since December 2003, we have completed five cases involving species identification and/or individualization of samples. One of our most complete datasets, in terms of statewide sampling coverage, is for elk. Our elk population is composed of 10 defined "herds" or management units, and includes the Roosevelt and Rocky Mountain subspecies. To better understand the geographic structure of these elk, we recently completed microsatellite and control region sequencing analyses; we present here some results as they relate to the forensic analysis of these herds. (Keywords: Washington, population genetics, geographic structure, elk, forensics)

 

Animal DNA Evidence in Crime-Scene Investigations
WICTUM, Elizabeth, and Sreetharan KANTHASWAMY
Veterinary Genetics Laboratory-Forensics, School of Veterinary Medicine, One Shields Avenue, University of California, Davis, California USA

Abstract: Due to the prevalence of domestic animals and their close relationships to humans, animal biological material is abundant and is frequently collected along with other evidence at crime-scene investigations. However, despite its potential as a tool in forensic investigations, this evidence remains largely underutilized by the law enforcement community. VGL Forensics is one of a handful of forensic laboratories dedicated to domestic animal DNA testing. DNA analyses of animal hair, urine, blood, saliva, feces, soft tissue, bones, and teeth have been used to successfully prosecute individuals by linking them to crime scenes in instances of burglary, sexual assault, arson, and murder as well as animal abuse and animal theft. (Keywords: DNA typing, animal, STR, mitochondrial, dog)

 

Track Evidence and Wildlife Investigations – More Than Boots and Truck Tires!
WOLFE, James
Alaska State Crime Lab, P.O. Box 210090, University of Alaska, Anchorage, Alaska 99521 USA

Abstract: Locating and collecting tiretrack and shoe impression evidence is an integral part of any crime scene investigation. Wildlife investigations often require the investigator to collect track evidence unique to the remote nature of many wildlife crime scenes. These tracks may include airplane tracks (both wheels and skis), ATV tracks (4-wheeler, 3-wheeler and rubber tracked) and snowmachine tracks. The collection and preservation of these tracks can present a challenge not only to the investigator at the crime scene, but also to the analyst at the crime lab who is faced with the task of performing a comparison on these tracks. The examiner has to devise techniques to obtain test impressions, and will also need to obtain information on the variation of given types of designs and vehicle construction in order to provide some background to help the jury determine the probative value of a given comparison. Examples of cases involving airplane and snowmachine tracks will be given, along with a discussion on techniques for documenting these tracks at the crime scene. (Keywords: Track evidence, crime)