Hindering the illegal trade in dog and cat furs through a DNA-based protocol for species identification

Abstract

In Western countries dogs and cats are the most popular pets, and people are increasingly opposed to their rearing for the fur industry. In 2007, a Regulation of the European Union (EU) banned the use and trade of dog and cat furs, but an official analytical protocol to identify them as source species was not provided, and violations of law are still frequent in all Member States. In this paper we report on the development and validation of a simple and affordable DNA method for species detection in furs to use as an effective tool to combat illegal trade in fur products. A set of mitochondrial primers was designed for amplification of partial cytochrome b, control region and ND1 gene in highly degraded samples, like furs and pelts. Our amplification workflow involved the use of a non-specific primer pair to perform a first test to identify the species through sequencing, then the application of species-specific primer pairs to use in singleplex end-point PCRs as confirmation tests. The advantage of this two-step procedure is twofold: on the one hand it minimises the possibility of negative test results from degraded samples, since failure of amplification with a first set of primers can be offset by successful amplification of the second, and on the other it adds confidence and reliability to final authentication of species. All designed primers were validated on a reference collection of tissue samples, obtaining solid results in terms of specificity, sensitivity, repeatability and reproducibility. Application of the protocol on real caseworks from seized furs yielded successful results also from old and dyed furs, suggesting that age and chemical staining do not necessarily affect positive amplifications. Major pros of this approach are: (1) sensitive and informative primer sets for detection of species; (2) short PCR amplicons for the analysis of poor quality DNA; (3) binding primers that avoid contamination from human DNA; (4) user-friendly protocol for any laboratory equipped for analysis of low-copy-number DNA. Our molecular procedure proved to be a good starting point for enforcing the EU Regulation against dog and cat fur trade in forensic contexts where source attribution is essential to the assignment of responsibilities.

A combined morphological and molecular approach for hair identification to comply with the European ban on dog and cat fur trade

“…Eventually, molecular species identification of the 21 “not excluded” samples led to ascertain the presence of C. lupus ssp. in five samples, which were probably illicit furs (Garofalo et al, 2018).…”

“…While DNA analysis is highly effective in fur identification at the species level (Garofalo et al, 2018), molecular testing is also expensive and labor-intensive, so it would not be reasonable to apply it arbitrarily to all the casework samples. Furthermore, in samples where no DNA amplification can be obtained, it is possible to exclude canid or felid species with a previous cheaper morphological screening.…”

“…The 25 casework samples were examined in parallel with both the morphological method and DNA analysis, to test the two approaches, with the ultimate goal of combining them in a diagnostic procedure aimed at discriminating dog and cat furs from allowed fur-bearing species. We followed a molecular method (Garofalo et al, 2018) aimed at targeting canid and felid mitochondrial DNA with specific primer pairs, that deliberately avoid binding to human DNA. Human contamination, coming from handling of furs during production and supply chain, can complicate the interpretation of results.…”

“…Human contamination, coming from handling of furs during production and supply chain, can complicate the interpretation of results. In brief, we applied a two-step DNA analysis that involved initially a nonspecific primer pair to identify the species through sequencing, then species-specific primer pairs to use in singleplex end-point PCRs for species confirmation as described by Garofalo et al (2018).…”

Objective discrimination of fur type based on electrophoresis optimized for fur‐hair proteins

“…As each species has its own characteristic DNA sequence, a DNA-based method was developed for the objective discrimination of the origin of fur [2,4]. In previous reports, animal-species-specific DNA sequences have been determined for the identification of the source animal by PCR.…”