Mycoplasma agassizii (Mycoplasmopsis agassizii) is a major pathogen responsible for upper respiratory tract disease (URTD) in tortoises, contributing to worldwide population declines. Despite its significance, tools for strain-level identification and epidemiological tracking remain limited. This study proposes a Multilocus Sequence Typing (MLST) scheme based on eight housekeeping genes (uvrB, tpiA, gyrB, efTu, rpoB, uvrA, gmK, and atpG), combined with Nanopore sequencing, to characterize M. agassizii isolates from tortoises in Italy and Portugal. Among 127 samples, 73 (57.5%) tested positive for M. agassizii. Analysis of 15 isolates revealed 14 distinct sequence types (STs). High genetic diversity was observed, even among samples from the same rescue centers. No correlation was found between STs and geographic origin or tortoise species. Four loci (uvrB, gyrB, rpoB, and gmK) were sufficient to discriminate all STs at a haplotype diversity cut-off ≥ 0.83. The standardized index of association (Ia = 0.2246) suggests a partially clonal population with evidence of recombination. This MLST scheme offers a promising tool for tracking M. agassizii strain diversity and understanding its epidemiology. Broader application across symptomatic and asymptomatic hosts is needed to evaluate associations between specific strains and disease outcomes, which will support conservation and URTD control efforts in tortoises.

The Sardinian wildcat (Felis silvestris lybica) belongs to a felid North-African lineage that reached the Mediterranean island in Neolithic times. While numerous studies have been conducted on the European wildcat (Felis silvestris silvestris), very little is known about the Sardinian wildcat ecology, genetics, and conservation status. Therefore, it is paramount to investigate the current distribution and genetics of the Sardinian population of F. s. lybica, previously described as var. sarda. In this work we assessed morphological and molecular traits of Sardinian wildcats, ascertained areas of species presence on the island, and investigated possible occurrence of hybridization with domestic cats. An integrated approach was adopted, involving questionnaire distribution and interviews to local people, and the morphological evaluation of road-killed individuals combined with their genetic typing using 29 polymorphic autosomal microsatellite loci. Genotypes were then analyzed by Bayesian clustering and assignment analyses. Questionnaires and interviews allowed us to build a preliminary alleged distribution map of the species and select candidate areas for non-invasive monitoring. The morphological and genetic characterization of road kills and museum specimens allowed us to identify pure individuals and recent wild × domestic hybrids, paving the way for better characterizing the Sardinian wildcat. Further steps of the research will allow us to shed light on this elusive presence on the island and enhance its conservation.

Island species are often understudied although being frequently represented by small and fragmented populations, potentially vulnerable to extinction. We investigated the phylogenetic position of hedgehogs living in Malta, using mitochondrial DNA control region analysis. A total of eleven Algerian hedgehog Atelerix algirus samples provided by a Wildlife Rescue Centre in Malta were processed for DNA extraction and sequencing. Phylogeographic analyses suggested the presence of different haplogroups within the species A. algirus: one endemic to Morocco (and the Canary Islands), another widely distributed in North Africa (reaching Spain and the Balearic Islands), and a third haplogroup represented by the two haplotypes detected in Malta, a diverging lineage typical of the island. We discuss management and conservation implications and put the basis for further research on Maltese hedgehogs.

Comparative landscape genetics studies provide insights on the impact of landscape elements on gene flow patterns of different species inhabiting the same geographic area. We investigated the population genetic structure of two sympatric ungulates, roe deer Capreolus capreolus and Northern chamois Rupicapra rupicapra, in a mountain area of the central Italian Alps (Trentino, northern Italy). A total of 122 chamois and 72 roe deer samples were genotyped by two species-specific panels of 11 polymorphic microsatellite loci and analyzed by aspatial and spatially explicit analyses. While the roe deer population resulted unstructured, a clear population structure was detected in chamois, with two main groups, one inhabiting the eastern and the other spread in the western part of the study area. Landscape genetics analysis confirmed these scenarios and revealed a different effect of landscape on gene flow. An IBD (Isolation-By-Distance) model best explained genetic variation in roe deer, while IBR (Isolation-By-Resistance) was found as the process underlying genetic variation patterns in chamois, suggesting arable lands, coniferous forests, watercourses, and main roads as potential barriers. Species distribution and landscape use might explain these results: roe deer mostly occupy valley floors relatively connected to each other, and their spatial behavior may promote gene flow across areas. On the other hand, chamois prefer higher elevations and their movements may be hindered by valleys, rivers, and road networks. This study highlights the different impacts of natural and anthropic landscape elements on gene flow in two sympatric species, resulting from their different ecological requirements.

After facing a great decline all over Europe during the past centuries, starting from the second half of the XX century the roe deer (Capreolus capreolus) was reintroduced and strongly managed throughout its range, as other ungulate species. Overhunting and habitat change were the main factors threatening roe deer populations in Italy, where small remnant populations of putatively native roe deer survived in a few localities of eastern Alps and central-southern Italy. We investigated the genetic variation of a roe deer population inhabiting the northern Apennines in the province of Massa-Carrara (Tuscany, Italy), analysing both mitochondrial DNA control region and a total of 11 autosomal microsatellite loci, to identify possible sources and recolonisation patterns, as well as the local prevalence of native Capreolus capreolus italicus gene pool. Analyses revealed an admixed nature of roe deer in this area, merging both native and non-native lineages, with a dominance of italicus haplotypes in the matriline and a majority of non-native genetic component in the autosomal markers. The high similarity with roe deer from neighbouring areas suggests a natural population origin by immigration. Two scenarios may explain the observed pattern of genetic variation: a colonisation by a limited number of immigrants from a single admixed source (either north or south-east), or a two-step recolonisation, firstly from the south, where the italicus ancestry was prevalent, and then from the north, mostly by individuals carrying C. c. capreolus genes. This study shows the genetic consequences of translocations even in populations not directly targeted by human interventions and highlight how investigating genetic variation might be essential in species management.

AbstractPatterns of genetic differentiation within and among animal populations might vary due to the simple effect of distance or landscape features hindering gene flow. An assessment of how landscape connectivity affects gene flow can help guide management, especially in fragmented landscapes. Our objective was to analyze population genetic structure and landscape genetics of the native wild boar (Sus scrofa meridionalis) population inhabiting the island of Sardinia (Italy), and test for the existence of Isolation‐by‐Distance (IBD), Isolation‐by‐Barrier (IBB), and Isolation‐by‐Resistance (IBR). A total of 393 Sardinian wild boar samples were analyzed using a set of 16 microsatellite loci. Signals of genetic introgression from introduced non‐native wild boars or from domestic pigs were revealed by a Bayesian cluster analysis including 250 reference individuals belonging to European wild populations and domestic breeds. After removal of introgressed individuals, genetic structure in the population was investigated by different statistical approaches, supporting a partition into five discrete subpopulations, corresponding to five geographic areas on the island: north‐west (NW), central west (CW), south‐west (SW), north‐central east (NCE), and south‐east (SE). To test the IBD, IBB, and IBR hypotheses, we optimized resistance surfaces using genetic algorithms and linear mixed‐effects models with a maximum likelihood population effects parameterization. Landscape genetics analyses revealed that genetic discontinuities between subpopulations can be explained by landscape elements, suggesting that main roads, urban settings, and intensively cultivated areas are hampering gene flow (and thus individual movements) within the Sardinian wild boar population. Our results reveal how human‐transformed landscapes can affect genetic connectivity even in a large‐sized and highly mobile mammal such as the wild boar, and provide crucial information to manage the spread of pathogens, including the African Swine Fever virus, endemic in Sardinia.

The limited knowledge on Papillomavirus diversity (particularly in wild animal species) influences the accuracy of PVs phylogeny and their evolutionary history, and hinders the comprehension of PVs pathogenicity, especially the mechanism of virus - related cancer progression. This study reports the identification of Leopardus wiedii Papillomavirus type 1 (LwiePV1), the first PV type within Lambdapapillomavirus in a Leopardus host. LwiePV1 full genome sequencing allowed the investigation of its taxonomic position and phylogeny. Based on results, LwiePV1 should be assigned to a novel PV species providing evidence for a polyphyletic origin of feline lambda PVs, and representing an exception to codivergence between feline lambda PVs and their hosts. Results improve our knowledge on PV diversity and pave the way to future studies investigating biological and evolutionary features of animal PVs.

Circoviruses are small circular DNA viruses causing severe pig and poultry disease, recently identified in various bat species worldwide. We report the detection and full-genome molecular characterization of a novel bat-associated Circovirus identified in faecal samples of Miniopterus schreibersii bats (Schreiber's bent-winged bats) from Sardinia, Italy. Full-genomic sequencing revealed a new putative member of Circoviridae family, with a genome size of 2063 nt. Sequencing allowed the characterization of the two major ORFs, inversely arranged, encoding replicase and capsid proteins, as well as the finding of a polythymidine tract within the genome, and highlighted phylogenetic relationships of the novel virus. This is the first report of circovirus in European bats. Giving the high level of genetic diversity of bat circoviruses, it is paramount to further investigate the relationships between these viruses and bats.

Poxvirus infections have been reported in domestic, captive, and wild avian hosts including many raptor species. A wild Common Buzzard (Buteo buteo) admitted to a wildlife veterinary clinic in Sardinia, Italy, showed multiple, wart-like proliferative cutaneous lesions on both legs. Histologically, there was ballooning degeneration and large intracytoplasmic inclusion bodies consistent with avipoxvirus (APV) infection. Diagnosis was confirmed by PCR detecting APV genes: P4b (locus fpv167), P35 (locus fpv140), and partial DNA polymerase. Phylogenetic analyses were performed to compare the detected virus with a panel of selected APVs. Analyses of P4b and DNA polymerase assigned the virus to clade A (fowlpox virus), subclade A7, grouping with many other APVs previously isolated in birds of prey. Further research should highlight the diversity of avian pox viral strains circulating among Common Buzzards as well as the phylogenetic role of locus fpv140 (P35) in comparison with the more-conserved P4b and DNA polymerase genes.

Bats may be natural reservoirs for a large variety of emerging viruses, including mammalian coronaviruses (CoV). The recent emergence of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV) in humans, with evidence that these viruses may have their ancestry in bats, highlights the importance of virus surveillance in bat populations. Here, we report the identification and molecular characterization of a bat β-Coronavirus, detected during a viral survey carried out on different bat species in the island of Sardinia (Italy). Cutaneous, oral swabs, and faecal samples were collected from 46 bats, belonging to 15 different species, and tested for viral presence. Coronavirus RNA was detected in faecal samples from three different species: the greater horseshoe bat ( Rhinolophus ferrumequinum ), the brown long-eared bat ( Plecotus auritus ), and the European free-tailed bat ( Tadarida teniotis ). Phylogenetic analyses based on RNA-dependent RNA polymerase (RdRp) sequences assigned the detected CoV to clade 2b within betacoronaviruses, clustering with SARS-like bat CoVs previously reported. These findings point to the need for continued surveillance of bat CoV circulating in Sardinian bats, and extend the current knowledge on CoV ecology with novel sequences detected in bat species not previously described as β-Coronavirus hosts.

Anaplasma species are globally distributed tick-borne bacteria causing a range of clinical conditions in domestic animals, wildlife, and human. Nevertheless, data on presence and distribution of Anaplasma strains in ticks are still lacking, especially in the Mediterranean region. This study reports the molecular identification, genetic characterization and phylogeny of Anaplasma strains of both veterinary and zoonotic importance in ticks collected from domestic and wild hosts sampled in a typical Mediterranean warm temperate region, the island of Sardinia. Results reveal the presence of A. ovis in Rhipicephalus bursa and R. sanguineus sensu lato ticks; A. platys-like was found in R. bursa ticks; while A. platys and A. phagocytophilum were detected in Hyalomma marginatum and H. lusitanicum ticks. Investigating the occurrence of different Anaplasma species in ticks from domestic and wild hosts improves the knowledge of tick-borne diseases in the Mediterranean area, and has implications in defining vector species distribution and risk assessment.

Avipoxvirus (APV) infections have been observed in a wide variety of wild, captive and domestic avian hosts, recently including a range of island endemic and endangered species. However, not enough is known about genome diversity and phylogenetic relationships of APVs, as well as their host-range specificity. A wild stone curlew ( Burhinus oedicnemus ) was recovered in Sardinia (Italy), showing large wart-like lesions and nodules on both legs and toes, which resulted positive to poxvirus by PCR. Histopathological examination of the lesions showed ballooning degeneration and large intracytoplasmic inclusion bodies consistent with APV infection. A multiple gene sequencing approach was applied to highlight the phylogenetic relationships of this virus with a panel of selected APVs at the clade and subclade levels. This novel isolate was characterized by sequencing partial 4b core protein, P35 (locus fpv140) and DNA polymerase genes and phylogenetic analyses assigned it to clade A, (Fowlpox virus, FWPV), subclade A2. Conservation implications of avian pox presence in Sardinian stone curlews and possibly in other island bird species are discussed.

Visual counts gathered within citizen science programs are increasingly used to determine distribution and abundance of species of conservation concern. However, to obtain reliable patterns from counts, imperfect detection should always be considered, with particular reference to rare and elusive species. By analysing data from a citizen science monitoring program based on multiple simultaneous observers, we studied detection probability of the Sardinian mountain newt, Euproctus platycephalus. Detectability of individual newts widely varied among observers, and was positively affected by the number of newts exposed to during sampling. Training, although appearing to improve detectability, did not accommodate for differences among trained observers. No effect of sampling hour, tree shade, cloud cover, water flow, turbidity, and temperature was found, possibly due to standardisation of sampling conditions. Depending on observer's skills and the population exposed to during sampling, detection probability of newt populations can widely vary. Most of the sampling units (pools) had few newts exposed to during sampling, with a high probability of recording false absences. Herpetological surveys could be more extensively based on multiple simultaneous observers to reduce observer heterogeneity bias in the detection process, and obtain more reliable patterns of species abundance and distribution for conservation purposes.

AbstractAssessing and updating the extinction risk and conservation status of species and populations is paramount to guide management strategies. Maintaining up-to-date and realistic geographical distribution maps of individual species is one aspect of this. We report an updated distribution for an Italian island endemic amphibian, the Sardinian newt Euproctus platycephalus, categorized as Endangered on the IUCN global and national Red Lists. The distribution of E. platycephalus was reassessed by means of visual surveys, questionnaires, interviews and scientific literature. The species was found over a geographical range comparable to that used for the IUCN assessment but we recorded a significantly larger number of populations (57 vs 14). There was no appreciable difference in the species’ Extent of Occurrence between 1972–1974 and 2010–2015. Area of Occupancy increased between past (1972–1974 and 1999–2000) and present (2010–2015) distribution records. Based on this updated distribution and considering that several new populations have been found, the distribution of the species may still be underestimated. Given the novel distribution data provided here and the need for new long-term demographic data, we recommend that the conservation status of E. platycephalus be reassessed. In the Italian national Red List a potential overstatement of extinction risk is evident for other amphibian species, possibly because the information used in their assessment is deficient. Considering that monitoring rare and elusive species is costly and time consuming we recommend more extensive use of multiple sources of information for Red List assessments.

Mycoplasma spp. have been detected in birds of prey, but their prevalence in free living raptors and their significance to birds' health need further investigation. Molecular techniques have been increasingly used to identify mycoplasmas in various avian species, due to the fastidious nature of these pathogens hampering traditional bacteriologic tests. This study reports the identification of 23 novel mycoplasma sequences during the monitoring of 62 birds of prey on admission to wildlife centers in Sardinia, Italy. Molecular investigation performed on pharyngeal swabs revealed 26 birds positive to Mycoplasma (42%). Sequence analysis based on 16S rRNA, 16S-23S rRNA intergenic spacer, and RNA polymerase β subunit (rpoB) gene highlighted cluster assignment and phylogenetic relationships among the identified types, classified within the hominis group. Additionally, Ornithobacterium rhinotracheale, associated with respiratory disease in poultry, was identified in 17 birds (27%). Potential coinfection and mycoplasma opportunistic nature present implications for raptor species conservation.

The recent characterization of the 18S ribosomal RNA (rRNA) of a pathogenic Babesia species in a domestic sow paved the way for establishing diagnostic and epidemiological tools for porcine babesiosis. Here, we developed the first specific Babesia sp. Suis PCR, and we applied this test to a panel of samples collected from animals living in a typical Mediterranean environment (Sardinia, Italy), including domestic pigs, wild boars, and ticks. In domestic pigs, PCR coupled with sequencing revealed an estimated Babesia infection frequency of 26.2% and the presence of distinct 18S sequence types. The different distribution of sequence types in symptomatic and asymptomatic subjects might suggest the existence of phylogenetically closely related strains with variable pathogenicity in pigs. Moreover, molecular identification of tick species indicated Rhipicephalus sanguineus and Rhipicephalus bursa as candidate vectors potentially involved in the transmission of this pathogen. Collectively, the data reveal the suitability of 18S rRNA PCR/sequencing for molecular diagnosis of porcine babesiosis and for large-scale investigations on the presence and geographical distribution of Babesia sp. Suis genetic variants.

Recently, herpes viruses have been detected in different cetacean species from the Atlantic and in Mediterranean striped dolphins (Stenella coeruleoalba). While pathogens such as cetacean morbillivirus have been widely studied following recent epizootics, herpesvirus (HV) distribution and pathogenic effects in cetaceans are still understudied. This study reports the first molecular identification of a Gammaherpesvirus in the genital mucosa of a free-living Mediterranean bottlenose dolphin (Tursiops truncatus) stranded off the coast of central Italy. Sequenced herpesviral PCR product was closely related to other HVs recently isolated in the genital mucosa of various cetacean species.

Free-living and captive chelonians might suffer from upper respiratory tract disease (URTD), a pathology primarily caused by Mycoplasma agassizii. Wild tortoises can also be an important reservoir of Salmonella spp., which are commensal in the host reptile but are potential zoonotic agents. Between July 2009 and June 2010, we screened free-living European tortoises (spur-thighed tortoises Testudo graeca, Hermann's tortoises Testudo hermanni, marginated tortoises Testudo marginata) temporarily housed in a wildlife center in Italy. We molecularly characterized 13 Mycoplasma isolates detected in all Testudo spp. studied, and three PCR-positive animals showed typical URTD clinical signs at the time of sampling. Three Salmonella enterica serotypes (Abony, Potsdam, Granlo), already related to reptile-associated human infections, were also identified. These results highlight the potential role played by wildlife recovery centers in the spread and transmission of pathogens among wild chelonians and to humans.

Papillomaviruses play an important role in human cancer development, and have been isolated from a number of animal malignancies. However, the association of papillomaviruses with tumors has been poorly investigated in sheep. In this study, a novel ovine Papillomavirus, OaPV3, was cloned from sheep squamous cell carcinoma. Unlike the already known ovine papillomaviruses, belonging to the Delta genus, OaPV3 lacks the E5 open reading frame and maintains the conserved retinoblastoma motif in the E7 gene. OaPV3 infects exclusively epithelial cells, and was found in skin of healthy sheep of geographically separated flocks located in Sardinia (Italy). This new virus is transcriptionally active in tumors and shares low homology with all the other papillomaviruses, establishing a new genus. Taken together, the co-occurrence of OaPV3 and tumors, its cell and tissue tropism, and its gene repertoire, suggests a role for this virus in development of sheep squamous cell carcinoma.