Species identification in routine casework samples using the SPInDel kit

The identification of species in casework samples is of fundamental importance for forensic investigations. Laboratories are increasingly compelled to provide accurate and fast identifications in trace materials left on crime scenes, wildlife poaching, illegal trade of protected species, fraudulent food products cases, etc. However, the field of nonhuman forensic genetics is still working on the standardization of typing methods and practices. Here we describe the successful implementation of the Species Identification by Insertions/Deletions (SPInDel) method in routine casework analyses in 11 laboratories worldwide. The SPInDel was developed to detect human DNA, at the same time that identifies common animal species. The fragment size analysis of six mtDNA regions allows identification in suboptimal DNA samples, including mixtures, with no need for sequencing. The samples were collected from 2013 to 2018 and included hair, blood, meat, saliva, faeces, bones, etc. The SPInDel kit successfully identified >95% of the samples, being dog, human and pig the most frequently detected species. The six SPInDel loci were successfully amplified in mixtures and degraded samples (river water, sand, stains in clothes, etc.). Interestingly, several species that were not originally targeted by SPInDel primers were also identified (e.g., red fox, brown bear, fallow deer and red deer). In conclusion, the SPInDel kit was successfully used in crime scene investigations (often involving human DNA detection) and in cases of poaching, environmental contamination and food fraud. It is now becoming a useful tool for the routine analysis of nonhuman DNA samples within the high quality standards of forensic genetics.     

DNA检验技术在野生动物保护中的应用

随着生物技术的发展,法医DNA检验技术在保护野生动物的工作中得到了越来越多的应用.本文介绍了全球野生动物犯罪现状及保护措施,简述了法医DNA检验技术在野生动物物种鉴定、来源地确认、个体识别和亲子鉴定等方面的实际应用,重点评述了法医DNA技术用于野生动物保护的几种常用检验方法以及各自的优缺点,探讨了法医DNA技术用于野生...     

Victimless venison? Deer poaching and black market meat in the UK

Abstract

The British Deer Society places the number of poached deer in the UK as high as 50,000 each year whereas only 335 incidents were reported to the police in 2009. This article explores deer as invisible victims of green crime and the motivations behind this poaching, drawing on the typologies of wildlife crime offenders developed by Nurse in 2013. In particular, the traditional profit-driven motivation of offenders is explored by attempting to uncover if there is, as suspected, a black market in venison. In order to do this, online questionnaires were sent to wildlife crime officers and gamekeepers asking questions about the scale and scope of deer poaching, who the perpetrators are and what is happening to the meat. From these data, it is hoped that a more detailed picture of deer poaching can be developed to further inform wildlife law and poaching prevention. This article also draws attention to the plight of poached deer and the potential danger to people of a black market in venison.     

Comparing Discourse to Officer Perceptions: The Problems of War and Militarization in Wildlife Crime Enforcement

‘War’ has become a common model and metaphor for biodiversity conservation in Africa. By discussing the specific challenges of wildlife crime enforcement in Uganda, this article challenges the ‘war on wildlife crime’ discourse. It concludes that in the context of Uganda, the discourse is profoundly unhelpful because of a lack of alignment between the problems highlighted by Ugandan law enforcement officers interviewed and the solutions typically favoured in the ‘wars on crime’. Most wildlife crimes are subsistence-driven and interviewees’ requests are for basic equipment and conventional capacity building. Findings demonstrate that the language of war, militarization and securitization should be used with caution as it risks constructing an image of wildlife crime that is misleading—and one that prevents responses that are effective in the long term.     

An overview of seized illegal wildlife entering the United States

The current study analyses seizures made at US ports of entry between 2003 and 2013, with the aim to identify concentrations of illegal wildlife imports into the United States. Findings show that 94% of species seized belong to six groups – mammals, molluscs, birds, reptiles, fish and coral – with mammals and reptiles making up more than half of all seizure incidents. Additionally, most seized wildlife is imported as leather products, medicinal products and as meat. The majority of seizures emanate from six countries, and illegal wildlife is primarily brought to the US via airline baggage. Temporal trends of wildlife seizures point to increases in the seizures of all groups of species, with the exception of birds. Based on these findings, we recommend using situational crime prevention techniques at US ports of entry to reduce opportunities that enable this trade.     

Humans Are Animals,Too: Critical Commonalities and Differences Between Human and Wildlife Forensic Genetics

Wildlife forensics has recently been recognized among the wide variety of forensic science disciplines. This review compares human and wildlife DNA forensics, which use the same genetic tools, but often for far different purposes. Human forensic genetics almost invariably attempts to identify individual perpetrators involved in a given crime. Wildlife forensics often determines whether a crime has occurred. In addition to techniques familiar in human laboratories, like individual matching with STRs, wildlife analysts may be asked to determine the taxonomic identity, geographic source, or sex of evidence items, or the familial relationships or minimum number of individuals among a group of samples. This review highlights the common questions, legal framework, databases, and similar validation requirements to foster understanding between disciplines. Based on this understanding, human and wildlife DNA practitioners may work together and learn from each other in order to elevate the discipline of forensic genetics.     

Recovery of human DNA profiles from poached deer remains: A feasibility study

Poaching is a crime that occurs worldwide and can be extremely difficult to investigate and prosecute due to the nature of the evidence available. If a species is protected by international legislation such as the Convention on International Trade in Endangered Species of Wild Fauna and Flora then simply possessing any part of that species is illegal. Previous studies have focused on the identification of endangered species in cases of potential poaching. Difficulties arise if the poached animal is not endangered. Species such as deer have hunting seasons whereby they can legally be hunted however poaching is the illegal take of deer, irrespective of season. Therefore, identification of deer alone has little probative value as samples could have originated from legal hunting activities in season. After a deer is hunted it is usual to remove the innards, head and lower limbs. The limbs are removed through manual force and represent a potential source of human touch DNA.We investigate the potential to recover and profile human autosomal DNA from poached deer remains. Samples from the legs of ten culled deer were obtained (40 in total) using minitapes. DNA from samples was extracted, quantified and amplified to determine if it would be possible to recover human STR profiles.Low quantification data led to the use of an extended PCR cycling protocol of 34 cycles. Samples from seven deer amplified, however some samples were excluded from further analysis due to ‘drop in’ alleles or the low level of successfully amplified loci. Samples from five deer could be further analysed and gave match probabilities ranging from 6.37 × 10^{− 3} to 9.53 × 10^{− 11}.This study demonstrates the potential of recovering human touch DNA from poached animal remains. There is the potential for this test to be used in relation to other species of poached remains or other types of wildlife crimes. This is the first time, to our knowledge, that human STR profiling has been successfully applied to touch DNA in regards to simulated wildlife crime.     

Wildlife Crime: The Problems of Enforcement

Environmental and wildlife crime appear recently to be benefitting from an increasing profile amongst those agencies tasked with their control, as well as receiving growing criminological attention. Despite this, those with responsibilities in this area report that it remains marginalised, receiving limited resources and suffering from a lack of political impetus to push such problems higher up the agenda. This is particularly so for those agencies, such as the police, that may be seen to have many more pressing objectives. This discussion paper considers the problems of relying on an enforcement approach to controlling such offences, taking, as an example, those activities that may be termed ‘wildlife crime’, focusing on the situation in England and Wales. Firstly, the legislative framework that criminalises harm or exploitation of wildlife is presented, alongside the main enforcement methods used. Next, the problems facing an enforcement approach are critically considered, the key issues being: under-resourcing and marginalisation, the large ‘dark-figure’ of wildlife crime, the possibility of corruption, the lack of seriousness with which such crimes are viewed, and the lack of deterrent effect. Finally, responses to the problems of enforcement are presented, categorised as either methods to improve enforcement or, as the author advocates, methods which are alternatives to enforcement (such as adopting a crime prevention approach). The paper concludes with suggestions for future research in this field.     

DNA barcoding as a tool for species identification in three forensic wildlife cases in South Africa

Poaching of wildlife animals for subsistence and commercial purposes has lead to population declines in Africa. In forensic cases, a need exists to identify the species of origin of carcasses, meat or blood. In the study presented here, the mitochondrial COI gene was sequenced to determine the species of unknown samples in three suspect South African forensic wildlife cases. In two cases the unknown samples were identified as originating from domestic cattle (Bos taurus) and in the third case the sample was identified as common reedbuck (Redunca arundinum). This is the first report of the COI sequence of common reedbuck. The study highlights the need for accurate wildlife reference material from each country in order to convict wildlife cases.     

Recovery of human DNA profiles from poached deer remains part 2: Improved recovery protocol without the need for LCN analysis

Although poaching is a common wildlife crime, the high and prohibitive cost of specialised animal testing means that many cases are left un-investigated. We previously described a novel approach to wildlife crime investigation that looked at the identification of human DNA on poached animal remains (Tobe, Govan and Welch, 2011). Human DNA was successfully isolated and amplified from simulated poaching incidents, however a low template protocol was required which made this method unsuitable for use in many laboratories. We now report on an optimised recovery and amplification protocol which removes the need for low template analysis.Samples from 10 deer (40 samples total — one from each leg) analysed in the original study were re-analysed in the current study with an additional 11 deer samples. Four samples analysed using Chelex did not show any results and a new method was devised whereby the available DNA was concentrated. By combining the DNA extracts from all tapings of the same deer remains followed by concentration, the recovered quantity of human DNA was found to be 29.5 pg ± 43.2 pg, 31 × greater than the previous study. The use of the Investigator Decaplex SE (QIAGEN) STR kit provided better results in the form of more complete profiles than did the AmpF?STR® SGM Plus® kit at 30 cycles (Applied Biosystems). Re-analysis of the samples from the initial study using the new, optimised protocol resulted in an average increase of 18% of recovered alleles. Over 17 samples, 71% of the samples analysed using the optimised protocol showed sufficient amplification for comparison to a reference profile and gave match probabilities ranging from 7.7690 × 10^? 05 to 2.2706 × 10^? 14.The removal of low template analysis means this optimised method provides evidence of high probative value and is suitable for immediate use in forensic laboratories. All methods and techniques used are standard and are compatible with current SOPs. As no high cost non-human DNA analysis is required the overall process is no more expensive than the investigation of other volume crime samples. The technique is suitable for immediate use in poaching incidents.     

Validation of presumptive tests for non-human blood using Kastle Meyer and Hemastix reagents

Kastle Meyer and Hemastix reagents are presumptive tests commonly used in forensic casework for the detection of blood, and their suitability has been reviewed in numerous publications. However, studies to date have focused on the validation of these tests on human blood alone, and no published work has looked at the sensitivity, specificity and effect on DNA analysis when using these reagents to presumptively test for animal blood. The aim of this study was to validate the two reagents for use with animal blood, and compare their performance in order to choose the best test based on the circumstances in wildlife crime investigation.The sensitivity, specificity, stability and robustness of the methods were assessed by experiments with dilutions of animal blood (from 1:4 to 1:65536) using direct and indirect (rub) tests, potential interfering substances, blood sources from different species and aged blood. The effects of the two reagents on subsequent DNA analysis were also investigated.During the direct tests, Kastle Meyer showed a higher sensitivity, detecting blood down to a dilution of 1:16,384, one order of magnitude lower than Hemastix. However during the rub test, Hemastix showed a higher sensitivity, detecting blood down to a dilution of 1:64 on porous materials while Kastle Meyer was positive only down to a dilution of 1:16. Moreover, when using the same swab for presumptive testing and DNA extraction, Hemastix testing allowed amplification of a sufficient amount of DNA for species identification at its limit of sensitivity on porous materials (1:64) while Kastle Meyer inhibited most amplification of DNA at its less sensitive limit of 1:16 dilution. On the other hand, Hemastix showed a much lower specificity, producing false positive results when exposed to tomato, potato, rust, avian uric acid, bleach and sink rot, while Kastle Meyer only produced a faint positive reaction from potato. Both tests performed equally well detecting fresh blood of different animal species. The stability test gave comparable results among the tests except for aged fish blood stains, where the Kastle Meyer test performed poorly.Owing to its ease of use, higher sensitivity, and lack of interference with downstream DNA analysis, and despite its reduced specificity compared to Kastle Meyer, the Hemastix method is more appropriate for use in wildlife crime investigations. Positive results would always be confirmed with DNA analysis and the low interference of the reagent will allow the use of a single swab for presumptive testing and DNA sampling.     

DNA typing in a cattle stealing case.

DNA profiling was used as probative evidence in a cattle stealing case. The carcasses of the dead animals were found from a report and a farmer recognized the remains as those corresponding to the stolen animals by the farm mark on the coat. Those remains were collected as reference samples. Meat pieces were sequestered from a butchery and then sent to our Laboratory by the Justice Department of Buenos Aires (Argentine) to perform a DNA comparative analysis with the reference. Matches were found between the evidences and the references, supporting the hypothesis that the meat pieces had been obtained from the stolen animals. The butcher was suspected of stealing animals but no direct incrimination had been made yet.     

Preventing Wildlife Crimes: Solutions That Can Overcome the ‘Tragedy of the Commons’

The 'tragedy of the commons' dilemma occurs when individuals working independently of one another, will overuse a common-property resource for short-term benefits while decimating the resource for long-term use (Hardin 1968). This is often found in the field of wildlife crimes where species become overexploited to increase short-term profits while endangering and eliminating a natural resource for future users. Wildlife crimes suffering from the ‘tragedy’ need to be prevented in order for species to avoid extinction while also conserving a natural resource that monetarily benefits numerous people and their respective communities. Current approaches to the illegal wildlife trade include implementing trade bans or regulatory schemes at the national and international level, yet their effectiveness of reducing the trade is unknown. Perhaps, a better approach in reducing the illegal wildlife trade is a combination of making it more difficult to poach (i.e. situational crime prevention) and incentivizing locals to abstain from poaching. This paper will first review the literature on wildlife crimes and then use a case study approach that will examine the literature on the illegal parrot trade, the market for wildlife skins, and over-fishing. Through these case studies, a comprehensive review of the problem will be detailed as well as innovative conservation solutions that show promise in reducing the poaching and exploitation of species. Amongst these solutions will be the use of situational crime prevention that has shown immediate reductions in crime when tailored towards highly-targeted areas and crimes.     

Application of plant DNA markers in forensic botany: genetic comparison of Quercus evidence leaves to crime scene trees using microsatellites

As highly polymorphic DNA markers become increasingly available for a wide range of plant and animal species, there will be increasing opportunities for applications to forensic investigations. To date, however, relatively few studies have reported using DNA profiles of non-human species to place suspects at or near crime scenes. Here we describe an investigation of a double homicide of a female and her near-term fetus. Leaf material taken from a suspect's vehicle was identified to be that of sand live oak, Quercus geminata, the same tree species that occurred near a shallow grave where the victims were found. Quercus-specific DNA microsatellites were used to genotype both dried and fresh material from trees located near the burial site and from the material taken from the suspect's car. Samples from the local population of Q. geminata were also collected and genotyped in order to demonstrate that genetic variation at four microsatellite loci was sufficient to assign leaves to an individual tree with high statistical certainty. The cumulative average probability of identity for these four loci was 2.06x10(-6). DNA was successfully obtained from the dried leaf material although PCR amplification was more difficult than amplification of DNA from fresh leaves. The DNA profiles of the dried leaves from the suspect's car did not match those of the trees near the crime scene. Although this investigation did not provide evidence that could be used against the suspect, it does demonstrate the potential for plant microsatellite markers providing physical evidence that links plant materials to live plants at or near crime scenes.     

Application of species-specific polymerase chain reaction in the forensic identification of tiger species

Globally, tigers are considered to be endangered, and are listed on Appendix I of CITES. A simple test, using a species-specific primer pair, was developed to identify tiger meat, faeces and dried skin, and provide forensic evidence of illegal wildlife trade. The specific fragment of mitochondrial cytochrome b gene was also successfully amplified from raw DNA products extracted from single tiger hairs. This PCR-based approach opens a new avenue to forensic identification of less-than-optimal samples.     

Organized crime or crime that is organized? The parrot trade in the neotropics

The illegal wildlife trade is one of the most profitable illegal industries in the world, only behind the trafficking of drugs, guns, and humans. Because of the relative ease in poaching wildlife combined with the possibility of high profit margins, many assume that organized crime is heavily linked to the trade. One group of species that is poached, trafficked and sold in illicit markets throughout the world are parrots. While some have claimed organized crime groups are involved in the trade, parrot experts contend there is no evidence of organized crime being involved. The purpose of this paper is to examine the structural organization of the illegal parrot trade in the neotropics to determine if the trade is driven by organized crime or if it is a simply a crime that is organized. The following study is based on 38 interviews with parrot poachers, middlemen, wildlife market sellers, and others knowledgeable on the trade in multiple cities within Bolivia and Peru to better understand the organization of the trade. The results garnered from these interviews do not support the notion that organized crime is involved in the illegal parrot trade in either country. The vast majority of participants are freelance operators where there appears to be no formal organization between or amongst those operating in the parrot trade. Implications of findings are discussed.     

Identification of shed or plucked origin of Indian peafowl (Pavo cristatus) tail feathers: preliminary findings

Indian Peafowl (Pavo cristatus) tail covert feathers were studied to investigate the difference between shed and plucked feathers in the context of wildlife offence cases involving the killing of the Indian national bird for the purpose of plucking feathers. Plucked feathers were distinguished from shed feathers by examining their roots under low magnification of a stereoscopic microscope. A chemical test to show the presence of blood on the roots of plucked feathers was used to corroborate the plucked origin of feathers.     

Planned complex suicide by self-poisoning and a manipulated blank revolver: remarkable findings due to multiple gunshot wounds and self-made wooden projectiles

A remarkable case of complex suicide with poisoning and multiple self-inflicted gunshot wounds to the head is reported. After ingestion of a liquid insecticide, the victim shot himself twice in the head, once in each temple. Self-manufactured wooden dowels were used as projectiles in combination with conventional blank cartridges. The dowels had been glued on top of the blank cartridges and expelled by propellant gases. Moreover, the blank revolver used had been extensively manipulated in a rarely observed manner. Several occlusive devices had been removed from the barrel and the cylinder chamber to enable the expulsion of the wooden projectiles. The investigation of the methods used and the circumstances found at the scene pointed towards a planned complex suicide. A remarkable case with unusual projectiles, i.e., wooden dowels, fired by an extensively manipulated blank gun is reported, emphasizing the importance of close collaboration between the police firearm laboratory and forensic pathology in practical casework.     

Evaluation of amylase testing as a tool for saliva screening of crime scene trace swabs

Amylase testing has been used as a presumptive test for crime scene saliva for over three decades, mainly to locate saliva stains on surfaces. We have developed a saliva screening application for crime scene trace swabs, utilising an amylase sensitive paper (Phadebas^® Forensic Press test). Positive results were obtained for all tested dried saliva stains (0.5–32 μL) with high or intermediate amylase activity (840 and 290 kU/L). Results were typically obtained within 5 min, and all samples that produced DNA profiles were positive. However, salivary amylase activities, as well as DNA concentrations, vary significantly between individuals. We show that there is no correlation between amylase activity and amount of DNA in fresh saliva. Even so, a positive amylase result indicates presence of saliva, and thereby presence of DNA. Amylase testing may be useful for screening in investigations where the number of DNA analyses is limited due to cost, e.g., in volume crime.