Federica Gabbianelli

Genomic improvement and sustainable breeding of Mediterranean buffalo are hampered by the lack of breed-specific genomic resources compared with bovine. To address this gap, we aimed to identify a comprehensive set of high-confidence single-nucleotide polymorphisms (SNPs) in Mediterranean buffalo and evaluate their informativeness across other buffalo populations. A total of 58 whole-genome sequencing samples, from three different sets, were merged through the integration of short- and long-read sequencing technologies. Variants, both unique and shared among datasets, were identified using a combination of bioinformatic tools to increase the reliability of the dataset. From these, over 11 million high-confidence biallelic SNPs were identified in the Mediterranean breed. The same SNPs were also tested in additional populations (other rivers and swamps) to evaluate their polymorphism and missingness. These results provide a robust genomic resource for Mediterranean buffalo, overcoming the limitations of bovine-derived genotyping tools available today. The identified SNPs dataset lays the basis for the development of a breed-specific SNP array, providing a dense and informative set of markers that could support cost-effective SNP chip development compared with existing arrays and sequencing technologies. This resource will facilitate more accurate genomic selection, precision breeding, and the conservation of genetic diversity in the Mediterranean buffalo population.

Papillomaviruses (PVs) are small, non-enveloped viruses, ubiquitous across the animal kingdom. PVs induce diverse forms of infection, such as cutaneous papillomas, genital papillomatosis, and carcinomas. During a survey on the fertility status of a mare, a novel Equus caballus PV (EcPV) has been identified using Next Generation Sequencing, and it was further confirmed with genome-walking PCR and Sanger sequencing. The complete circular genome 7607 bp long shares 67% average percentage of identity with EcPV9, EcPV2, EcPV1, and EcPV6, justifying a new classification as Equus caballus PV 10 (EcPV10). All EcPV genes are conserved in EcPV10, and phylogenetic analysis indicates that EcPV10 is closely related to EcPV9 and EcPV2, genus Dyoiota 1. A preliminary EcPV10 genoprevalence study, carried out on 216 horses using Real Time PCRs, suggested a low incidence of this isolate (3.7%) compared to EcPVs of the same genus such as EcPV2 and EcPV9 in the same horse population. We hypothesize a transmission mechanism different from the one observed in the closely related EcPV9 and EcPV2 that particularly infect Thoroughbreds. This horse breed is usually submitted to natural mating, thus indicating a possible sexual diffusion. No differences were detected for breeds in terms of susceptibility to EcPV10. Further studies are needed to investigate the molecular mechanisms behind the host and EcPV10 infection to explain the reduced viral spread.

Mycobacterium avium ssp. paratuberculosis (MAP) is the causative agent of paratuberculosis (PTB), a widespread chronic enteritis of ruminants. The progression of the infection depends on the containment action of innate and cell-mediated immunity (CMI), and it is related to environmental and genetic factors. In particular, PTB susceptibility seems to be associated with specific genes coding for immune regulators involved in the cell-mediated response during the infection. The aim of this preliminary study was to verify, in Italian beef cattle, an association between MAP infectious status and the presence of single nucleotide polymorphisms (SNPs) in candidate genes. To the best of our knowledge, this is the first investigation conducted on a native beef cattle breed, known as Marchigiana, reared in Central Italy. The present research, based on a longitudinal study, aimed to identify and correlate phenotypic and genetic profiles characteristic of the subjects potentially able to contrast or contain PTB. In a MAP-infected herd, ELISA, IFN-γ tests, qPCR, and cultures were performed at a follow-up, occurring within a period ranging from three to six years, to evaluate the individual state of infection. Animals testing positive for at least one test were considered infected. DNA samples of 112 bovines, with known MAP statuses, were analyzed to verify an association with SNPs in the genes encoding gamma-interferon (BoIFNG), interleukin receptor 10 (IL10RA), interleukin receptor 12 (IL12RB2), and toll-like receptors (TLR1, TLR2, TLR4). Regarding statistical analysis, the differences among target genes and pairs of alleles in the analyzed groups of animals, were evaluated at a significance level of p < 0.05. For IL10RA and for IL12RB2 genes, relevant differences in genotypic frequencies among the considered cattle groups were observed. For all candidate genes studied in this investigation, SNP genotypes already associated with PTB resistance were found more frequently in our population, suggesting potential resistance traits in the Marchigiana breed.

Papillomavirus (PV) infections may be related to anogenital lesions and cancer development in humans and several other animal species. To date, 11 different PVs have been reported in horses. Among them, a newly described PV named Equus caballus Papillomavirus Type9 (EcPV9) was thus far only reported in the semen of a stallion with penile lesions in Australia. This study reports for the first time the presence of EcPV9 in asymptomatic Italian horses. From July 2020 to January 2022, genital brush samples were collected from 209 horses with no apparent signs of neoplastic disease and no PV-associated lesions, clinically examined at the Didactic Veterinary University Hospital (OVUD) of Perugia and at the Veterinary University Hospital (OVU) of Turin. Brushes were submitted to real-time PCR targeting the EcPV9-L1 region. The first amplification targeted a region of ~116 bp, followed by the amplification and sequencing of ~533 bp of the positive samples. EcPV9-L1 DNA was found in eleven horses (5.3%), all female and mainly English Thoroughbred. Co-infection with EcPV2-L1 was found in 7 out of the 11 EcPV9-L1 positive horses (63.6%). This study contributes to the description of the prevalence of exposure or infection of EcPVs in the horse population in Italy, for which data are still limited. In this regard, here we provide a phylogenetic analysis and the completely reconstructed viral genomes of two Italian EcPV type 9 isolates, as well as four EcPV type 2 obtained from co-infected animals.

The study of plant and animal remains from archaeological sites provides important evidence about past human diets and habits: this includes species selection, food preparation, consumption and disposal practices. Furthermore, such information may also provide inferences about social status. Data from refuse disposal features identified in some elite contexts in central Italy – a high-status residence in Celleno Vecchio (Viterbo) and the Baglioni-Santacroce castle in Graffignano (Viterbo), both in northern Latium, as well as the Santi Quattro Coronati ecclesiastical complex in Rome – allow to explore, using archaeobotanical, archaeozoological and genetic data, some of the different ways in which people expressed wealth by means of food during a period between the late Middle Ages and Renaissance.

The Cinta senese is a pig breed, highly esteemed for its meat and derived products, characterized by a black coat with a typical white “belt” and documented by scant iconography, since the 13^th–14^th century in Italy. A piece of pottery showing a Cinta pig was found in the Graffignano castle (Northern Latium, Italy) dated 15th–16th centuries, spurring us to investigate the diet of the inhabitants. Ancient DNA analysis was carried out on 21 pig specimens on three nuclear SNPs: (1) g.43597545C>T, on the KIT gene, informative for the identification of the Cinta senese breed; (2) rs81460129, on an intergenic region in chr. 16, which discriminates between domestic pigs and wild boars, and; (3) a SNP on the ZFY/ZFX homologous genes, to determine the sex of the individuals. Our results indicate that the Cinta senese was present in Northern Latium in Late Medieval time, although it was not the only breed, and that pigs, including Cinta, interbred with wild boars, suggesting free-range breeding for all types of pigs. Moreover, the unexpected high proportion of young females may be considered as evidence for the wealth of the family inhabiting the castle.

Summary: The thyroid‐stimulating hormone receptor (TSHR) has been indicated as a putative domestication gene in chicken. Comparison of WGS identified a variant in residue 558 of the transmembrane domain (TM) of TSHR, where the domestic chicken (GGD) presents an arginine, whereas the red jungle fowl (RJF) shares a conserved glycine with other vertebrates. This variant has been demonstrated to be associated with phenotypes that are important for domestication and related to thyroid regulation, such as less fearful behavior, reduced aggressive behavior and reduced dependence on seasonal reproduction in GGD as compared with RJF. By means of molecular dynamics simulations, we highlighted the structural and dynamic differences of variant Gly558Arg in the TSHR TM domain. Alterations in TM helix flexibility, structure and protein overall motion are described. The so‐called ‘arginine snorkeling’ of residue 568 in GGD is observed and we hypothesize it as the originating force that produces the observed whole‐protein perturbation in the helix bundle dynamics, capable of altering the TSHR signal transduction. The results are discussed in the context of their implications for a better understanding of biological mechanisms in chicken under control of the thyroid, such as body metabolism, as well as for their usefulness in biomedical research.