One health surveillance strategy for coronaviruses in Italian wildlife.

The recent reinforcement of CoV surveillance in animals fuelled by the COVID-19 pandemic provided increasing evidence that mammals other than bats might hide further diversity and play critical roles in human infectious diseases. This work describes the results of a two-year survey carried out in Italy with the double objective of uncovering CoV diversity associated with wildlife and of excluding the establishment of a reservoir for SARS-CoV-2 in particularly susceptible or exposed species. The survey targeted hosts from five different orders and was harmonised across the country in terms of sample size, target tissues, and molecular test. Results showed the circulation of 8 CoV species in 13 hosts out of the 42 screened. Coronaviruses were either typical of the host species/genus or normally associated with their domestic counterpart. Two novel viruses likely belonging to a novel CoV genus were found in mustelids. All samples were negative for SARS-CoV-2, with minimum detectable prevalence ranging between 0.49% and 4.78% in the 13 species reaching our threshold sample size of 59 individuals. Considering that within-species transmission in white-tailed deer resulted in raising the prevalence from 5% to 81% within a few months, this result would exclude a sustained cycle after spillback in the tested species.

A forensic genetic investigation reveals a captive origin for a wild alien population of raccoons in Italy.

Invasive alien species have extensively impacted the ecosystems, where they may affect the native biodiversity. The North American raccoon Procyon lotor is one of the most successful invaders in Europe since its introduction in the early twentieth century. In Italy, a wild population was first established in the North at the beginning of the 2000s following a local introduction event. A further self-sustaining population was reported ten years later in Central Italy. To support an official investigation by the authorities, who suspected a captive origin of the free-ranging raccoons in Central Italy, we used nuclear and mitochondrial DNA markers, combined with different statistical approaches, to characterise their gene pool and trace the source of the founders. Results revealed that founders came from a private zoo-park from which they had inadvertently escaped, soon establishing a reproductive population in the wild. Additionally, our mitochondrial DNA data were used to supplement the haplotype variability known to date in captive and wild raccoons from Europe, Asia and their native range. The comparisons allowed us to update previous networks based on the control region with a new mitochondrial lineage, which had not been detected so far.

Genetic distinctiveness of an endangered falcon: Implications for conservation in Europe.

In the Falconidae, the genus Falco comprises species of large birds of prey with wide distribution worldwide. However, the European lanner falcon Falco biarmicus feldeggii is rapidly heading for global extinction following a dramatic decline caused by anthropogenic interference. Conservation projects are currently underway with the main purpose of increasing its population size in the Mediterranean basin through captive breeding and release of birds into the wild. To support the projects, and strengthen the legitimacy of conservation efforts consistently with the Evolutionary Significant Unit concept, we explored the possibility of characterising the gene pool of the European lanner and reliably distinguishing it from other falcon taxa inhabiting the Mediterranean area, which show morphological and genetic similarities. To address the issue, we examined genetic variability at the nuclear level through the analysis of 12 neutral Short Tandem Repeat loci, and, for the first time in these taxa, two single-copy functional genes, coding for the brain-derived neurotrophic factor precursor and the oocyte maturation factor, respectively. The second exon of the major histocompatibility complex class II B gene was also investigated. Additionally, to frame our data with previously published data, we assess variation at the mitochondrial level by sequencing portions of the cytochrome b, 12S rRNA gene, and the control region. Our results showed that the European lanner is highly distinct from other falcon taxa, as revealed by nuclear, but not by mitochondrial DNA. We discuss our findings focusing on their implications for the preservation of this highly endangered European bird, and highlighted the critical role of genetic information in planning and monitoring concrete interventions.

Seroconversion of a Swine Herd in a Free-Range Rural Multi-Species Farm against HPAI H5N1 2.3.4.4b Clade Virus.

Starting from October 2021, several outbreaks of highly pathogenic avian influenza virus (HPAIV) subtype H5N1 were reported in wild and domestic birds in Italy. Following the detection of an HPAIV in a free-ranging poultry farm in Ostia, province of Rome, despite the lack of clinical signs, additional virological and serological analyses were conducted on samples collected from free-ranging pigs, reared in the same holding, due to their direct contact with the infected poultry. While the swine nasal swabs were all RT-PCR negative for the influenza type A matrix (M) gene, the majority (%) of the tested pigs resulted serologically positive for the hemagglutination inhibition test and microneutralization assay, using an H5N1 strain considered to be homologous to the virus detected in the farm. These results provide further evidence of the worrisome replicative fitness that HPAI H5Nx viruses of the 2.3.4.4b clade have in mammalian species. Moreover, our report calls for additional active surveillance, to promptly intercept occasional spillover transmissions to domestic mammals in close contact with HPAI affected birds. Strengthened biosecurity measures and efficient separation should be prioritized in mixed-species farms in areas at risk of HPAI introduction.

Genetic structure of pastoral and farmer populations in the African Sahel.

Traditional pastoralists survive in few places in the world. They can still be encountered in the African Sahel, where annual alternations of dry and wet seasons force them to continual mobility. Little is known about the genetic structure of these populations. We present here the population distribution of 312 hypervariable segment I mitochondrial DNA (mtDNA) and 364 Y-short tandem repeat haplotypes in both farmer and pastoralist groups from the Lake Chad Basin and the West African Sahel. We show that the majority of pastoral populations (represented in the African Sahel by the Fulani nomads) fail to show significant departure from neutrality for mtDNA as evidenced by Fu's Fs statistics and exhibit lower levels of intrapopulation diversity measures for mtDNA when contrasted with farmers. These differences were not observed for the Y chromosome. Furthermore, analyses of molecular variance and population distributions of the mtDNA haplotypes show more heterogeneity in the sedentary groups than in the pastoralists. On the other hand, pastoralists retain a signature of a wide phylogenetic distance contributing to their male gene pool, whereas in at least some of the farmer populations, a founder effect and/or drift might have led to the presence of a single major lineage. Interestingly, these observations are in contrast with those recorded in Central Asia, where similar comparisons of farmer and pastoral groups have recently been carried out. We can conclude that in Africa, there have been no substantial mating exchanges between the Fulani pastoralists coming to the Lake Chad Basin from the West African Sahel and their farmer neighbors. At the same time, we suggest that the emergence of pastoralism might be an earlier and/or a demographically more important event than the introduction of sedentary agriculture, at least in this part of Africa.

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.

Cardiovascular flukes (Trematoda: Spirorchiidae) in Caretta caretta Linnaeus, 1758 from the Mediterranean Sea.

BACKGROUND: The northern Adriatic Sea represents one of the most important neritic foraging grounds for the loggerhead sea turtle Caretta caretta L. in the Mediterranean Sea. Four genera of blood flukes with variable prevalence and pathogenic impact have been reported worldwide in this species. Hapalotrema Looss, 1899 and Amphiorchis Price, 1934 are the only two genera reported in Mediterranean waters; however, updated data describing spirorchiidiasis in the central and eastern Mediterranean and infection prevalence are still lacking. This work aimed to investigate the presence and pathology of spirorchiidiasis in C. caretta in the Mediterranean Sea. METHODS: One hundred sixty-eight animals stranded along the northwestern Adriatic coast between 2009 and 2015 were submitted to necropsy and subsequent analyses for the detection of adult flukes, detection of eggs in the faeces and spleen and histopathology. Molecular analyses were carried out on hosts (mitochondrial D-loop) and parasites (28S gene and ITS2 spacer) to trace the turtle origins and identify the fluke phylogenetic relationships. RESULTS: Spirorchiidiasis was detected in 16.7% of the animals. Hapalotrema mistroides (Monticelli, 1899) and Neospirorchis sp. were found in twenty-six and ten cases, respectively. Adult flukes were found in six cases, while eggs were detectable through copromicroscopic examination for all infected turtles, and the results for the detection of eggs in the spleen agreed with the copromicroscopic analysis. Only mild lesions were observed. Eggs of types 1 and 3 were grossly visible in the gastrointestinal mucosa, vasculitis was rarely observed in the heart and great vessels, and multifocal granulomas were widespread in the tissues. Molecular identification unambiguously assigned the spirorchiid samples to H. mistroides and Neospirorchis sp. Genetic characterization of loggerhead mtDNA pointed to a Mediterranean origin of the turtle hosts. CONCLUSION: This survey provides new data on the spread of spirorchiidiasis in the Mediterranean loggerhead sea turtle population and reports for the first time the presence of Neospirorchis spp. in this basin. The infections did not have a causal effect on the death nor a strong impact on the general health status of the animals.

Polymorphism in Mitochondrial Coding Regions of Mediterranean Loggerhead Turtles: Evolutionary Relevance and Structural Effects.

We sequenced coding portions (1.6 kb) of the mtDNA in 170 loggerhead (Caretta caretta) turtles sampled in the central Mediterranean. The sequences spanned the entire ND1 and ND3 genes, the tRNAGly and tRNAArg, plus the 3' and 5' termini of COXIII and ND4L genes, respectively. Based on our sequencing results and published complete mitogenomes, we constructed a maximum parsimony phylogeny of C. caretta matrilines that sheds new light on the evolutionary relationships within the collection of lineages found in the Mediterranean and so far recognized by D-loop haplotypes only. We show that the new variants are useful to understand the ancestry of extant haplotypes, to improve genetically based studies on the philopatry and migratory behavior of the species, and for conservation purposes. In order to better understand the biological significance of the observed variation, we addressed intraspecific nonsynonymous substitutions in the context of the three-dimensional modeled structures of ND1 and ND3. The positions of variant amino acids within the folded subunits are consistent with a coadaptation with the restructuring of membrane thickness, fluidity, and lipid composition, a well-known response mechanism to thermal conditions. The pattern of amino acid substitutions departs from neutrality, suggesting local adaptation and/or polymorphism-based local selection.