Wildlife can serve as a potential reservoir and spreader of resistant and pathogenic bacteria. Raptors, occupying the ecological position of apex or mesopredators, integrate exposure across different habitats and therefore serve as bioindicators of environmental dissemination of pathogens. In this study, we isolated 54 Escherichia coli (E. coli) strains from feces sampled from 64 raptors admitted to a wildlife rescue center in Central Italy. Phenotypic antimicrobial susceptibility testing was conducted, followed by molecular screening for resistance genes. Additionally, the presence of intestinal E. coli pathotypes, including STEC, EHEC, EPEC, ETEC, EAEC, EIEC, and NTEC, was evaluated through virulence gene analysis. Results indicated notable resistance to commonly used antimicrobials, with the highest percentages observed for ampicillin (40.74%), fluoroquinolones (31.48%), and tetracycline (25.93%). Molecular analysis of phenotypically resistant isolates identified the presence of several resistance genes, including blaTEM (13 isolates), blaCTX-M (4 isolates), blaCMY-2 (3 isolates), blaSHV (1 isolate), tet(A) (9 isolates), tet(B) (4 isolates), cat1 (1 isolate), and cmlA (2 isolates). Furthermore, 29.63% of isolates were classified as multidrug-resistant (MDR) and 7.41% as extensively drug-resistant (XDR). Regarding virulence profiles, one isolate harboring eaeA, escV, and astA genes was classified as atypical EPEC, while 27.78% isolates had only the astA gene, preventing precise pathotype assignment. These findings highlight the circulation of antimicrobial-resistant and potentially pathogenic E. coli strains within raptor populations in Central Italy, emphasizing the zoonotic potential and reaffirming the role of raptors as bioindicators within a One Health approach.

Background/Objectives: European hedgehogs (Erinaceus europaeus) are present in areas where there is human activity; therefore, they can be a source of pathogens for other animals and humans. Methods: Eighteen hedgehog carcasses were collected and analyzed for Staphylococcus spp. Isolated strains were typed and analyzed for exfoliative toxins genes and the phenotypic and genotypic characteristics of antimicrobial resistance. Results: A total of 54 strains were isolated and typed as S. aureus, S. xylosus, S. sciuri, S. pseudintermedius, S. simulans, S. chromogenes, S. epidermidis, S. hyicus, and S. lentus. No strains had the eta and etb genes coding for exfoliative toxins. Overall, 39/54 (72.20%) isolates showed phenotypic resistance to at least one antimicrobial and 21/54 (38.80%) showed more than one resistance. The lowest efficacy was observed for erythromycin, with 40/54 (74.08%) strains classified as intermediate and 6/54 (11.11%) classified as resistant. Among the 29 isolates shown to be penicillin-resistant, 11 (37.93%) were oxacillin-resistant, with a minimum inhibitory concentration (MIC). Among the 54 staphylococcal strains, 2 (3.70%) were resistant to vancomycin, both with an MIC value equal to the maximum concentration of the antibiotic tested (256 μg/mL) and 2 (3.70%) had an intermediate resistance profile with an 8 μg/mL MIC value. No strains had the genes vanA and vanB. Two of the 29 (6.90%) penicillin-resistant strains had the blaZ gene; 8 (27.13%) strains had the mecA gene. Overall, 2/54 (3.70%) isolates were classified as extensively drug-resistant (XDR) and 9/54 (16.66%) were classified as multidrug-resistant (MDR). Conclusions: Hedgehogs can harbor antimicrobial-resistant staphylococci and can be sources of these bacteria for other animals and humans. They can also serve as bioindicators of the pathogens and antimicrobial-resistant bacteria circulating in a given habitat.

Reighardia sternae is a pentastomid parasitising the respiratory system of larids and skuas, the pathological significance of which is still poorly understood. In this study, pentastomes found in the interclavicular air sac of a deceased and necropsied yellow-legged gull (Larus michahellis) were identified by parasitological and molecular methods and associated gross and histopathological lesions were evaluated. After morphological evaluation and molecular characterisation, the species R. sternae was identified. This is the first report of R. sternae infecting L. michahellis in Italy. Gross pathology showed generalised aerosacculitis, parabronchial edema, and enlargement of the heart, liver, and spleen. The systemic lesions found at histopathological examination were indicative of chronic cardiocirculatory failure, which led to the death of the gull. Moreover, inflammation was present throughout the lungs and air sacs in the form of some granulomas and diffuse small nodular aggregates rich in lymphocytes. The remnants of a parasite were observed within a granulomatous lesion in the lung, suggesting a primary host response to the parasite. These latter findings may suggest that the migration of R. sternae was the cause of the pathological granulomatous pictures in the examined yellow-legged gull.

Birds are known to be carriers of ticks infected by tick-borne pathogens, including bacteria. However, not many studies have been carried out on avian tissues to detect these agents. The aim of the present survey was to investigate, using PCR, the presence of Anaplasma phagocytophilum, Bartonella spp., Borrelia burgdorferi sensu lato, Chlamydia psittaci, Coxiella burnetii, Ehrlichia canis, Francisella tularensis, and Rickettsia spp. in the spleens collected from 300 wild birds of different orders and species from Central Italy. A total of 53 (17.67%) samples were PCR positive for at least one investigated pathogen. One (0.33%) bird was positive for Bartonella spp., five (1.67%) birds were positive for C. burnetii, eleven (3.67%) for B. burgdorferi s.l., and thirty-six (12%) for C. psittaci. No coinfection was detected. All samples were negative for A. phagocytophilum, E. canis, F. tularensis, and Rickettsia spp. The findings showed that wild birds may harbor different zoonotic tick-borne bacteria; therefore, they can contribute to the diffusion of these agents.

Seagulls are synanthropic wild birds that can contaminate, through their droppings, beaches, urban and peri-urban environments. This concern is more serious when seagulls eliminate antimicrobial-resistant pathogenic bacteria. This study analyzed the fecal samples from 137 yellow-legged seagulls (Larus michahellis) from Central Italy. A total of 218 Escherichia coli strains were isolated and analyzed for phenotypic and genotypic antimicrobial resistance and to identify the virulence genes characterizing different pathotypes. The disk diffusion method on all isolates found relevant resistance rates to ampicillin (38.99%), tetracycline (23.85%), and enrofloxacin (21.10%). On the basis of all results obtained with this test, 62 (28.44%) isolates were classified as multidrug-resistant (MDR) and 6 (2.75%) as extensive drug-resistant (XDR). Molecular analyses conducted on the strains phenotypically resistant to carbapenems, cephalosporins, and penicillins found 9/37 (24.32%) strains positive for blaOXA-48, 52/103 (50.49%) for blaTEM, 12/103 (11.65%) for blaCMY2, 3/103 (2.91%) for blaCTX, and 1/103 (0.97%,) for blaSHV. PCR to detect virulence genes characterizing different pathotypes found that 40 (18.35%) isolates had the astA gene, indicative of the enteroaggregative (EAEC) pathotype, 2 (0.92%) had cnf1, 2 (0.92%) had cnf2, and 1 (0.46%) had cdt-IV. All five (2.29%) strains were reportable as necrotoxigenic (NTEC), while 4 (1.83%) had both eaeA and escV, reportable as enteropathogenic (EPEC). Measures to limit seagulls’ access where humans and other animals reside are pivotal to reduce the risk of infection with antimicrobial-resistant and pathogenetic E. coli strains.

Perineuronal or neuronal satellitosis is the term describing the presence of glial cells in the satellite space surrounding the neuronal perikaryon. Confusingly, this finding has been described both as a physiologic and pathologic condition in humans and animals. In animals, neuronal satellitosis has been described in mammals, as well as in avian species. For the latter, the authors wondered whether neuronal satellitosis is expressed in the normal telencephalon of different avian orders and families and whether this pattern in different species shows a specific brain-region association. For these aims, this study explored the presence of neuronal satellitosis in the major areas of the healthy telencephalon in wild and domestic avian species of different orders and families, evaluating its grade in different brain regions. Neuronal satellitosis was seen in the hyperpallium and mesopallium as areas with the highest grade. Passeriformes showed the highest grade of neuronal satellitosis compared to diurnal or nocturnal raptors, and Charadriiformes. To clarify the exact role of neuronal satellitosis in animals without neurological disease, further studies are needed.RESEARCH HIGHLIGHTSNeuronal satellitosis is a common finding in the healthy avian telencephalon.Neuronal satellitosis is a species- and brain-region-associated finding in birds.Passeriformes have the highest grade of neuronal satellitosis.

Avipoxviruses (APVs) are important pathogens of both domestic and wild birds. The associated disease is characterized by skin proliferative lesions in the cutaneous form or by lesions of the first digestive and respiratory tracts in the diphtheritic form. Previous studies investigated these infections in symptomatic wild birds worldwide, including Italy, but data about the circulation of APVs in healthy avian wildlife are not available. The present study tested spleen samples from 300 wild birds without typical lesions to detect Avipoxvirus DNA. Overall, 43.33% of the samples scored positive. Aquatic birds were more frequently infected (55.42%) than other animals (26.40%), and in Anseriformes, high positivity was found (52.87%). The obtained results suggest that wild birds could be asymptomatic carriers of Avipoxviruses, opening new possible epidemiological scenarios.

Bacteria of the genus Enterococcus are opportunistic pathogens, part of the normal intestinal microflora of animals, able to acquire and transfer antimicrobial resistance genes. The aim of this study was to evaluate the possible role of wild avifauna as a source of antimicrobial-resistant enterococci. To assess this purpose, 103 Enterococcus spp. strains were isolated from the feces of wild birds of different species; they were tested for antimicrobial resistance against 21 molecules, vancomycin resistance, and high-level aminoglycosides resistance (HLAR). Furthermore, genes responsible for vancomycin, tetracycline, and HLAR were searched. E. faecium was the most frequently detected species (60.20% of isolates), followed by E. faecalis (34.95% of isolates). Overall, 99.02% of the isolated enterococci were classified as multidrug-resistant, with 19.41% extensively drug-resistant, and 2.91% possible pan drug-resistant strains. Most of the isolates were susceptible to amoxicillin/clavulanic acid (77.67%) and ampicillin (75.73%), with only 5.83% of isolates showing an ampicillin MIC ≥ 64 mg/L. HLAR was detected in 35.92% of isolates, mainly associated with the genes ant(6)-Ia and aac(6′)-Ie-aph(2″)-Ia. Few strains (4.85%) were resistant to vancomycin, and the genes vanA and vanB were not detected. A percentage of 54.37% of isolates showed resistance to tetracycline; tet(M) was the most frequently detected gene in these strains. Wild birds may contribute to the spreading of antimicrobial-resistant enterococci, which can affect other animals and humans. Constant monitoring is essential to face up to the evolving antimicrobial resistance issue, and monitoring programs should include wild avifauna, too.

The combined actions of climatic variations and landscape barriers shape the history of natural populations. When organisms follow their shifting niches, obstacles in the landscape can lead to the splitting of populations, on which evolution will then act independently. When two such populations are reunited, secondary contact occurs in a broad range of admixture patterns, from narrow hybrid zones to the complete dissolution of lineages. A previous study suggested that barn owls colonized the Western Palearctic after the last glaciation in a ring-like fashion around the Mediterranean Sea, and conjectured an admixture zone in the Balkans. Here, we take advantage of whole-genome sequences of 94 individuals across the Western Palearctic to reveal the complex history of the species in the region using observational and modeling approaches. Even though our results confirm that two distinct lineages colonized the region, one in Europe and one in the Levant, they suggest that it predates the last glaciation and identify a narrow secondary contact zone between the two in Anatolia. Nonetheless, we also show that barn owls re-colonized Europe after the glaciation from two distinct glacial refugia: a western one in Iberia and an eastern one in Italy. Both glacial lineages now communicate via eastern Europe, in a wide and permeable contact zone. This complex history of populations enlightens the taxonomy of Tyto alba in the region, highlights the key role played by mountain ranges and large water bodies as barriers and illustrates the power of population genomics in uncovering intricate demographic patterns.

We report an outbreak of canine distemper virus (CDV) among stone martens (Martes foina) in Italy. After being rescued in Northern Italy between April and June 2018, six subjects were kept in a wildlife and exotic animal rescue center in Bologna province. Subjects have been monitored for 15 months in captivity. Within this timelapse, two subjects died, while among the remaining four, only one showed clinical symptoms referable to distemper. Surviving subjects have been regularly tested for CDV by means of reverse transcriptase-PCR from conjunctival and oropharyngeal swabs for eleven months. The identified viruses belonged to the Wildlife-Europe CDV genetic subgroup. Neutralizing antibodies were detected at the end of the eleven months, when all subjects tested reverse transcriptase-PCR negative. Our findings confirm the circulation of the Wildlife-Europe CDV genetic subgroup (Europe 1/South America 1 lineage) within the Italian wildlife, and improve knowledge on viral infection in stone martens.

Two Eurasian Blackbirds (Turdus merula) from central Italy were found with severe cyclocoelid trematodosis associated with airsacculitis. The birds were submitted with severe respiratory distress; one died shortly after hospitalization, while the second bird was euthanized. At necropsy, a massive presence of cyclocoelid flukes was observed in the coelomic cavity and air sacs of both birds. The air sacs were diffusely opaque, thickened, and covered by scant fibrinous exudate mixed with numerous parasites. Histologically, the air sacs showed diffuse and severe oedema with fibrinous exudate. Diffuse mononucleated and heterophilic infiltration mixed with multiple granulomas contained degenerated trematodes. Morishitium polonicum was identified using morphologic keys and molecular analysis of extracted DNA. Infections caused by M. polonicum are poorly documented in blackbirds and the findings in these birds support the pathogenic role of this trematode as a potential cause of death in blackbirds in Italy. Extended epidemiologic surveys are required to properly assess the potential importance of M. polonicum as a life-threatening pathogen in Blackbird populations.

The study of insular populations was key in the development of evolutionary theory. The successful colonisation of an island depends on the geographic context, and specific characteristics of the organism and the island, but also on stochastic processes. As a result, apparently identical islands may harbour populations with contrasting histories. Here, we use whole genome sequences of 65 barn owls to investigate the patterns of inbreeding and genetic diversity of insular populations in the eastern Mediterranean Sea. We focus on Crete and Cyprus, islands with similar size, climate and distance to mainland, that provide natural replicates for a comparative analysis of the impacts of microevolutionary processes on isolated populations. We show that barn owl populations from each island have a separate origin, Crete being genetically more similar to other Greek islands and mainland Greece, and Cyprus more similar to the Levant. Further, our data show that their respective demographic histories following colonisation were also distinct. On the one hand, Crete harbours a small population and maintains very low levels of gene flow with neighbouring populations. This has resulted in low genetic diversity, strong genetic drift, increased relatedness in the population and remote inbreeding. Cyprus, on the other hand, appears to maintain enough gene flow with the mainland to avoid such an outcome. Our work provides a comparative population genomic analysis of the effects of neutral processes on a classical island-mainland model system. It provides empirical evidence for the role of stochastic processes in determining the fate of diverging isolated populations.

The combined actions of climatic variations and landscape barriers shape the history of natural populations. When organisms follow their shifting niches, obstacles in the landscape can lead to the splitting of populations, on which evolution will then act independently. When two such populations are reunited, secondary contact occurs in a broad range of admixture patterns, from narrow hybrid zones to the complete dissolution of lineages. A previous study suggested that barn owls colonized the Western Palearctic after the last glaciation in a ring-like fashion around the Mediterranean Sea, and conjectured an admixture zone in the Balkans. Here, we take advantage of whole-genome sequences of 94 individuals across the Western Palearctic to reveal the complex history of the species in the region using observational and modeling approaches. Even though our results confirm that two distinct lineages colonized the region, one in Europe and one in the Levant, they suggest that it predates the last glaciation and identify a secondary contact zone between the two in Anatolia. We also show that barn owls recolonized Europe after the glaciation from two distinct glacial refugia: a previously identified western one in Iberia and a new eastern one in Italy. Both glacial lineages now communicate via eastern Europe, in a wide and permeable contact zone. This complex history of populations enlightens the taxonomy of Tyto alba in the region, highlights the key role played by mountain ranges and large water bodies as barriers and illustrates the power of population genomics in uncovering intricate demographic patterns.

The Leucocytozoon genus comprises numerous widely distributed parasites which have been less investigated than other avian hemoprotozoa. Their occurrence is common, with very variable prevalence values and pathogenicity degrees. Leucocytozoon species are characterized by a great taxonomic diversity, and infections are usually restricted to birds of the same family. In the present paper, a mixed hemosporidia infection by Leucocytozoon californicus and Haemoproteus sp. in an adult male merlin (Falco columbarius) which died during hospitalisation is reported, indicating, for the first time, a newly described avian host species. A molecular investigation was carried out through cytochrome b gene analysis, revealing a 100% match with L. californicus and Haemoproteus spp. A blood smear examination allowed us to detect Leucocytozoon fusiform mature gametocytes and different degrees of maturity of Haemoproteus gametocytes. Histopathology revealed foci of necrosis, hemorrhagic areas and extramedullary hematopoiesis in the liver, the presence of microthrombi in the heart and lung and scattered hemorrhages in the lung.

The role of avian species in the Neospora caninum life cycle has not been completely elucidated, and epidemiological data from Europe are scant. The aim of the present report was to evaluate the presence of N. caninum DNA in the tissues of 302 birds belonging to different avian species, along with IFAT titers. Forty-two out of the 302 birds (13.9%) showed low serological titers (1/50 and 1/100) against N. caninum. These data, positive for 31 animals (10.3%), were corroborated by PCR. Twenty-two hearts and eighteen brains scored positive, while nine subjects resulted had parasite DNA both in their hearts and brains. Serological data showed significantly higher results in waterfowl in respect to non-waterfowl avian species. This finding indicates a higher exposure of water birds to the parasite. These avian species, in fact, which feed directly from soil and/or water, are prone to ingest oocysts excreted by final canid hosts. The present study adds information to the state of art of N. caninum epidemiology in Italy, even if more investigations using bio-assays are needed to allow for a serological/parasitological follow-up to evaluate the real impact of the avian species in maintaining the parasite in main reservoirs.

Objective: To investigate the potential role of wild birds as fecal spreaders of enteropathogenic,enterohemorrhagic and Shiga-toxins producing Escherichia coli（E. coli）,enteropathogenic E. coli,enterohemorrhagic E. coli and Shiga toxin-producing E. coli strains. Methods: Fecal samples collected from 121 wild birds of different orders and species were submitted to molecular analyses. In particular,eaeA encoding intimin,hlyA encoding for hemolysin,stx1 and stx2 genes encoding Shiga-toxins 1 and 2,respectively,were investigated. Results: Overall,21（17.35%） fecal samples resulted positive for at least one of the investigated genes. In detail,12（9.91%） samples were positive for eaeA,10（8.26%） for stx1,4（3.31%） for hylA and 1（0.83%） for stx2. An owl（Athene noctua） positive for the four investigated genes suggesting that it harbored a STEC strain. However,virulence genes characterizing EPEC,and EHEC strains were mainly found among seagulls,waterfowl and feral pigeons. Conclusions: Seagulls,waterfowl and feral pigeons,which frequently reach and contaminate rural,urban and peri-urban areas with their droppings,may be important sources of E. coli infection for other animals and humans.

Toxoplasma gondii is an obligate apicomplexan zoonotic parasite that infects humans and other animals and is responsible for toxoplasmosis. This parasite causes one of the most common parasitic infections in humans worldwide. Toxoplasmosis meets the requirements for a One Health Disease due to its ability to affect the health of human beings as well as domestic and free ranging animals. Integrating human, domestic animal, and wildlife data could better assess the risk and devise methods of control. A first step of such an approach would be the knowledge of the prevalence of parasitosis in humans and animals in selected areas. Therefore, the aim of the present study was to evaluate the occurrence of Toxoplasma infection in 216 free ranging birds belonging to different genera/species by serology and molecular techniques. Twenty-five out of 216 animals (11.6%) were positive to the immunofluorescence antibody test (IFAT) with antibody titers ranging from 1/20 to 1/320, and 19 of them (8.8%) also showed a positive PCR for Toxoplasma DNA. The results confirmed the widespread occurrence of Toxoplasma infection in wild birds and serological data were corroborated by molecular results in birds that also had low antibody titers. The knowledge of the wide occurrence of the parasite in game and wild birds should enhance the accurate estimation of the risks in handling, managing, and eating these species with regard to domestic carnivores as well as the impact of viscera and offal in the environment.