Date Published: June 14, 2018
Publisher: Public Library of Science
Author(s): Kyle M. Ewart, Greta J. Frankham, Ross McEwing, Dang Tat The, Carolyn J. Hogg, Claire Wade, Nathan Lo, Rebecca N. Johnson, Ruslan Kalendar.
Rhinoceros (rhinos) have suffered a dramatic increase in poaching over the past decade due to the growing demand for rhino horn products in Asia. One way to reverse this trend is to enhance enforcement and intelligence gathering tools used for species identification of horns, in particular making them fast, inexpensive and accurate. Traditionally, species identification tests are based on DNA sequence data, which, depending on laboratory resources, can be either time or cost prohibitive. This study presents a rapid rhino species identification test, utilizing species-specific primers within the cytochrome b gene multiplexed in a single reaction, with a presumptive species identification based on the length of the resultant amplicon. This multiplex PCR assay can provide a presumptive species identification result in less than 24 hours. Sequence-based definitive testing can be conducted if/when required (e.g. court purposes). This work also presents an actual casework scenario in which the presumptive test was successfully utlitised, in concert with sequence-based definitive testing. The test was carried out on seized suspected rhino horns tested at the Institute of Ecology and Biological Resources, the CITES mandated laboratory in Vietnam, a country that is known to be a major source of demand for rhino horns. This test represents the basis for which future ‘rapid species identification tests’ can be trialed.
The illegal wildlife trade is a multi-billion dollar transnational industry that constitutes one of the top five forms of black market activities . Wildlife forensics is an important tool for wildlife law enforcement and managing wildlife trade activities. DNA-based wildlife forensic science is an evolving discipline which combines techniques utilized by conservation genetics and human forensic science, and their application in the legal system . Technological advancements and the copious genomic sequence data now available offer the potential to improve wildlife forensic techniques and hence enforcement . However, countries with limited financial resources and/or infrastructure capacity may have difficulties to consistently carry out DNA-based forensic testing, especially when faced with numerous large seizures. Additionally, in some juristictions there may be strict time constraints on intelligence gathering or statutes of limitations. The development of rapid and cost-effective wildlife forensics techniques is therefore vital for laboratories that are subject to such constraints, so that they may increase conviction rates and improve enforcement outcomes.
This newly designed multiplex PCR assay protocol, illustrated in Fig 1, is able to provide a presumptive species identification for black rhino, white rhino and Indian rhino in less than 24 hours on the basis of an amplicon length that is species-specific.
This work presents the first fast and accurate presumptive species identification test and casework data developed for one of the highest profile groups of animals involved in illegal wildlife trafficking, the rhinos’. The multiplex PCR assay described here significantly reduces the material and time costs of previous rhino horn species identification methods, whilst still providing a robust and accurate presumptive species identification for rhino horn. It is imperative that laboratories in Asian countries, where demand for these illegal products is currently highest, perform species identification testing to contribute to enforcement actions around horn traficking crimes and to monitor market trends [6, 7]. However, these laboratories may have limited capacity to produce timley and robust identifications. Currently, validated testing relies on Sanger sequencing  which may not be feasible for a number of reasons including a lack of labor, funding, infrastructure and/or expertise. This is an urgent concern for Vietnam, because the time required for transfer of material to the laboratory, DNA extraction and PCR amplification, and sequencing (which often takes weeks at IEBR) can hinder the chance of a successful prosecution. Our multiplex PCR assay was successfully implemented and field-tested at IEBR, by their own laboratory staff, and can now be used to facilitate timely and robust presumptive identifications for rhino horn trafficking investigations in Vietnam, allowing rapid and reliable information to flow back to enforcement agencies. Addtionally, it allows for testing of horns, to ensure animal disease regulations are met prior to sending samples to better equiped or specialised laboratories, such as the South African RhODIS laboratory .
We are currently amidst a rhino poaching crisis, in which over 1000 rhinos are being poached each year [4, 15]. The multiplex PCR assay presented here provides an effective means to generate presumptive species identifications for rhino horn from commonly traded species within 24 hours of samples arriving at a laboratory. This assay will increase the speed and reduce the material and time costs of the current species identification techniques that rely on sequencing and sequence analysis by allowing for more efficient triaging of suspected horn products for further downstream analysis and potential enforcement. Increasing the capacity to conduct rhino horn investigations, particularly for ‘front-line’ laboratories such as IEBR, is a vital component to combat the trade [4, 18]. It is important to consistently identify the species of seized horn products, not only to assist in securing a conviction, but also to monitor the market trends of rhino horn trafficking in range state and destination countries.