Characterization of prevalence and genetic subtypes of Blastocystis sp. in wild and domestic Suidae of central Italy aided by amplicon NGS.

Blastocystis spp. is a common single-celled intestinal symbiont, comprising several genetic subtypes (ST) and transmissible by animal-to-animal, human-to-human, animal-to-human and, possibly, human-to-animal routes. This work was designed to explore the presence of Blastocystis in sympatric domestic and wild suids and their ability to carry zoonotic STs, in a condition of widespread opportunity to come in contact with the microorganism through their shared water and food resources, and other carriers. We sampled 42 and 37 stool samples from wild boars and domestic pigs, respectively. STs were first identified by PCR followed by Sanger sequencing. Sequences represented in double-band PCR products or in Sanger chromatograms displaying multiple peaks, were resolved by next generation sequencing (NGS). Twenty-six (61.9%) wild boar and 26 (70.2%) pig samples were PCR-positive, respectively. ST3, ST5 and ST15 were found in 3.8%, 38.4% and 80.8% of the positive wild boars and 11.5%, 88.5%, 11.5% of the positive pigs, respectively. ST1 was found only in pigs (3.8%). STs 5 and 15 were common in both groups of animals, but in reversed proportions, suggesting preferential colonization. We found significantly different ST distributions among wild boars and domestic pigs. This might indicate that lifestyle differences between the two populations influence their risk for contracting certain subtypes, or that ST5 and ST15 can colonize preferentially wild or domestic animals. Based on the STs described here, wild boars and domestic pigs can act as reservoirs with zoonotic potential. The ability of suids to carry zoonotic STs appears to be higher when using NGS than Sanger sequencing, and resolution of complex sequencing profiles is imperative before excluding the presence of STs of human concern.

Matching STR and SNP genotyping to discriminate between wild boar, domestic pigs and their recent hybrids for forensic purposes.

The genetic discrimination between phylogenetically close taxa can be challenging if their gene pools are not differentiated and there are many shared polymorphisms. The gene flow between wild boar (Sus scrofa) and domestic pig (S. s. domesticus) has never been interrupted from domestication onwards, due to non-stop natural and human-mediated crossbreeding. To date there are no individual genetic markers that are able to distinguish between the two forms, nor even to identify effectively their hybrids. We developed a combined molecular protocol based on multiplex porcine-specific STR-profiling system and new real time PCR-based assays of single polymorphisms in the NR6A1 and MC1R genes to gain high diagnostic power in the differentiation of wild boar, pig and hybrids for forensic purposes. The combined approach correctly assigned individuals to one or the other parental gene pool and identified admixed genotypes. Evidence was found for substantial reduction of false negative results by using multiple marker systems jointly, compared to their use individually. Our protocol is a powerful and cost-effective diagnostic tool that can easily be adopted by most forensic laboratories to assist authorities contrast food adulteration, assure veterinary public health and fight against wildlife crimes, like poaching and illegal detention of wild animals.

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

Animal furs are encountering more and more the detriment of public opinion, that is increasingly sensitive to animals, their welfare and protection. The feeling of outrage against animal suffering is particularly intense when cats and dogs are involved, since these are the most popular pets in Western countries. However, in some Asian countries breeding of dogs and cats for the fur industry is a common practice. These furs and their finished garments are often mislabelled in order to be imported and sold to unaware consumers in Western countries. The European Union has issued the Regulation 1523/2007, which bans the use and trade of dog and cat furs. The main purposes of the Regulation were to normalise the internal market and to address the concerns of European consumers about the risk of inadvertently buying products containing these species. The Regulation states that several analytical methods (microscopy, DNA testing and mass spectrometry) can be used to exclude dogs and cats as source species, but an official analytical protocol was not provided. In this paper, we report on the development of a reliable and affordable method for species identification in furs, based on a combined morphological and molecular approach. Our protocol provides an initial morphological analysis as a time and cost effective screening test. Only samples that are morphologically not excluded as canid/felid furs, based on few selected microscopic features, are then submitted to DNA testing. The application of this protocol on seized furs reached 92% identification of species. Our approach assists in identifying frauds and reinforcing the ban on dog and cat fur trade, allowing (1) rapid inexpensive recognition of fake furs, (2) exclusion of non-canid/non-felid furs through fast microscopic morphological screening, (3) overall cost reduction with lower number of samples to be submitted to DNA analysis, (4) analytical protocol to stand in court in case criminal sanctions are to be applied.

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

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.

Wildlife molecular forensics: identification of the Sardinian mouflon using STR profiling and the Bayesian assignment test.

A forensic short tandem repeat (STR) typing test using a population database was developed to investigate an instance of poaching on the protected Sardinian mouflon. The case study involves a suspected poacher found in possession of a carcass, which he claimed was that of a sheep from his flock and had died accidentally. His claim was refuted by the molecular forensic analyses as DNA typing and the Bayesian assignment test revealed the carcass to be mouflon-derived; the genetic profile of the carcass matched also that of additional trace evidence collected by forestry officers at the scene of the kill. The matching evidence led to the poacher being charged with the illegal harvest of protected wildlife. Molecular techniques, in combination with a reference population database, and the appropriate statistical evaluation of genetic information, are fundamental to wildlife forensics. This approach allows DNA testing to be accepted in court as submissible evidence in the fight against poaching and other crimes involving wildlife.