Are Parapoxvirus zoonotic diseases doomed to remain neglected?

Parapoxvirus (PPV) infections are considered neglected zoonoses because their incidence is often unknown or greatly underestimated despite being endemic globally. Here, we report the comprehensive diagnostic workflow that led to the identification of two cases of persistent PPV infections. The results obtained underline the importance of adopting a "One Health" approach and cross-sectoral collaboration between human and veterinary medicine for precise aetiological diagnosis and correct management of patients affected by zoonotic diseases.

Eating the Enemy: Mycoplasma Strategies to Evade Neutrophil Extracellular Traps (NETs) Promoting Bacterial Nucleotides Uptake and Inflammatory Damage.

Neutrophils are effector cells involved in the innate immune response against infection; they kill infectious agents in the intracellular compartment (phagocytosis) or in the extracellular milieu (degranulation). Moreover, neutrophils release neutrophil extracellular traps (NETs), complex structures composed of a scaffold of decondensed DNA associated with histones and antimicrobial compounds; NETs entrap infectious agents, preventing their spread and promoting their clearance. NET formation is triggered by microbial compounds, but many microorganisms have evolved several strategies for NET evasion. In addition, the dysregulated production of NETs is associated with chronic inflammatory diseases. Mycoplasmas are reduced genome bacteria, able to induce chronic infections with recurrent inflammatory symptoms. Mycoplasmas' parasitic lifestyle relies on metabolite uptake from the host. Mycoplasmas induce NET release, but their surface or secreted nucleases digest the NETs' DNA scaffold, allowing them to escape from entrapment and providing essential nucleotide precursors, thus promoting the infection. The presence of Mycoplasma species has been associated with chronic inflammatory disorders, such as systemic lupus erythematosus, rheumatoid arthritis, inflammatory bowel disease, Crohn's disease, and cancer. The persistence of mycoplasma infection and prolonged NET release may contribute to the onset of chronic inflammatory diseases and needs further investigation and insights.

MHO_0730 as a Surface-Exposed Calcium-Dependent Nuclease of Mycoplasma hominis Promoting Neutrophil Extracellular Trap Formation and Escape.

Mycoplasma lipoproteins play a relevant role in pathogenicity and directly interact with the host immune system. Among human mycoplasmas, Mycoplasma hominis is described as a commensal bacterium that can be associated with a number of genital and extragenital conditions. Mechanisms of M. hominis pathogenicity are still largely obscure, and only a limited number of proteins have been associated with virulence. The current study focused on investigating the role of MHO_0730 as a virulence factor and demonstrated that MHO_0730 is a surface lipoprotein, potentially expressed in vivo during natural infection, acting both as a nuclease with its amino acidic portion and as a potent inducer of Neutrophil extracellular trapsosis with its N-terminal lipid moiety. Evidence for M. hominis neutrophil extracellular trap escape is also presented. Results highlight the relevance of MHO_0730 in promoting infection and modulation and evasion of innate immunity and provide additional knowledge on M. hominis virulence and survival in the host.

Characterization of paucibacillary ileal lesions in sheep with subclinical active infection by Mycobacterium avium subsp. paratuberculosis.

Paratuberculosis (PTB) or Johne's disease is a contagious enteritis of ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP). Ovine PTB is less understood than bovine PTB, especially concerning paucibacillary infection and its evolution into clinical disease. We combined shotgun proteomics, histopathology and immunohistochemistry for the characterization of ileal tissues collected from seven asymptomatic sheep negative to serum ELISA, positive to feces and tissue MAP IS900 and F57 PCR, histologically classified as paucibacillary, actively infected, together with 3 MAP-free controls (K). Following shotgun proteomics with label-free quantitation and differential analysis, 96 proteins were significantly changed in PTB vs K, and were mostly involved in immune defense processes and in the macrophage-MAP interaction. Principal component analysis (PCA) of protein abundances highlighted two PTB sample clusters, PTB1 and PTB2, indicating a dichotomy in their proteomic profiles. This was in line with the PCA of histopathology data and was related to features of type 2 (PTB1) and type 3a (PTB2) lesions, respectively. PTB2 proteomes differed more than PTB1 proteomes from K: 43 proteins changed significantly only in PTB2 and 11 only in PTB1. The differential proteins cathelicidin, haptoglobin, S100A8 and S100A9 were evaluated by immunohistochemistry. K tissues were negative to cathelicidin and haptoglobin and sparsely positive to S100A8 and S100A9. PTB tissues were positive to all four proteins, with significantly more cells in PTB2 than in PTB1. In conclusion, we described several pathways altered in paucibacillary PTB, highlighted some proteomic differences among paucibacillary PTB cases, and identified potential markers for disease understanding, staging, and detection.

Mycoplasma lipoproteins are major determinants of neutrophil extracellular trap formation.

Neutrophil granulocytes are paramount to innate responses as major effectors of acute inflammation. Among the various strategies enacted by neutrophils to eliminate microbes NETosis is a novel distinct antimicrobial activity in which an interlacement of chromatin fibres rich in granule-derived antimicrobial peptides and enzymes is extruded (NETs, neutrophils extracellular traps ). NETs contribute to the pathogenesis of acute and chronic inflammatory disorders. The interactions of mycoplasmas and innate immune cells, in particular neutrophil granulocytes, are poorly defined. Here, we describe NET formation in vivo in the mammary gland and milk of sheep naturally infected by Mycoplasma agalactiae. Also, we assess the contribution of liposoluble proteins, the most abundant component of the Mycoplasma membrane, in inducing NETosis. We demonstrate that Mycoplasma liposoluble proteins induce NET release at levels comparable to what observed with other stimuli, such as lipopolysaccharides and phorbol 12-myristate 13-acetate. Stimulation of neutrophils with synthetic diacylated lipopeptides based on the M. agalactiae P48, P80, and MAG_1000 proteins, combined in a mix or used individually, suggests that NETosis might not be dependent on a specific lipopeptide sequence. Also, NETosis is partially abolished when TLR2 is blocked with specific antibodies. The results presented in this work provide evidences for the mechanisms underlying NET activation in mycoplasma infections, and on their contribution to pathogenesis of mycoplasmosis.

Neutrophil extracellular traps in sheep mastitis.

Neutrophil extracellular traps (NETs) are structures composed of DNA, histones, and antimicrobial proteins that are released extracellularly by neutrophils and other immune cells as a means for trapping and killing invading pathogens. Here, we describe NET formation in milk and in mammary alveoli of mastitic sheep, and provide a dataset of proteins found in association to these structures. Nucleic acid staining, immunomicroscopy and fluorescent in-situ hybridization of mastitic mammary tissue from sheep infected with Streptococcus uberis demonstrated the presence of extranuclear DNA colocalizing with antimicrobial proteins, histones, and bacteria. Then, proteomic analysis by LTQ-Orbitrap Velos mass spectrometry provided detailed information on protein abundance changes occurring in milk upon infection. As a result, 1095 unique proteins were identified, of which 287 being significantly more abundant in mastitic milk. Upon protein ontology classification, the most represented localization classes for upregulated proteins were the cytoplasmic granule, the nucleus, and the mitochondrion, while function classes were mostly related to immune defence and inflammation pathways. All known NET markers were massively increased, including histones, granule proteases, and antimicrobial proteins. Of note was the detection of protein arginine deiminases (PAD3 and PAD4). These enzymes are responsible for citrullination, the post-translational modification that is known to trigger NET formation by inducing chromatin decondensation and extracellular release of NETs. As a further observation, citrullinated residues were detected by tandem mass spectrometry in histones of samples from mastitic animals. In conclusion, this work provides novel microscopic and proteomic information on NETs formed in vivo in the mammary gland, and reports the most complete database of proteins increased in milk upon bacterial mastitis.

Ovine papillomaviruses: Diversity, pathogenicity, and evolution.

The family Papillomaviridae includes a plethora of viral species infecting virtually all vertebrates excluding amphibians, with astonishing impact on human and animal health. Although more than 250 species have been described in humans, the total number of papillomaviruses (PVs) discovered in animals does not reach up to this number. In animals, PV infections are mostly asymptomatic or can cause variable clinical conditions ranging from self-limiting papillomas and other cutaneous and mucosal benign lesions to cancer. Most of animal PV types have been discovered in cattle, dogs, horses, and cats with other farm host species remaining overlooked. In particular, the number of PV types so far identified in sheep is limited. This paper comprehensively reviews ovine PVs features, including viral taxonomy and evolution; genome organization; viral tropism and pathogenesis; macroscopical features and histopathological patterns, as well as available diagnostics tools. Data are critically presented and discussed in terms of impact on veterinary and public health. The development of future dedicated research is also discussed.

Ovine papillomavirus type 3 virus-like particle-based tools for diagnosis and detection of infection.

The design of prophylactic and diagnostic tools specific to animal papillomaviruses is hampered by the difficulties of viral in vitro manipulation and by the scarce availability of dedicated biotechnological tools. This paper reports the production of Ovine Papillomavirus 3 (OaPV3)-based virus-like particles (OaPV3-VLPs) in the baculovirus system and their use to investigate host humoral immune response through the establishment of an indirect ELISA test., Polyclonal sera and monoclonal antibodies were generated against OaPV3-VLPs, and their isotype and reactivity were determined. Additionally, antibodies allowed OaPV3 detection in ovine squamous cell carcinoma (SCC) samples by immunohistochemistry. Results encourage the standardization of OaPV3-specific prophylactic and serological diagnostic tools, and open new perspectives for the study of host-viral interaction and SCC development.

The Global Evolutionary History of Orf Virus in Sheep and Goats Revealed by Whole Genomes Data.

Orf virus (ORFV) belongs to the genus Parapoxvirus (Poxviridae family). It is the causative agent of contagious ecthyma (CE) that is an economically detrimental disease affecting small ruminants globally. Contagious ecthyma outbreaks are usually reported in intensive breeding of sheep and goats but they have also been reported in wildlife species. Notably, ORFV can infect humans, leading to a zoonotic disease. This study aims to elucidate the global evolutionary history of ORFV genomes in sheep and goats, including the first genomes from Central America in the analyses. In comparison to the last study on ORFV whole genomes, the database now includes 11 more sheep and goat genomes, representing an increase of 42%. The analysis of such a broader database made it possible to obtain a fine molecular dating of the coalescent time for ORFV S and G genomes, further highlighting the genetic structuring between sheep and goat genomes and corroborating their emergence in the latter half of 20th century.

Production and release of antimicrobial and immune defense proteins by mammary epithelial cells following Streptococcus uberis infection of sheep.

Investigating the innate immune response mediators released in milk has manifold implications, spanning from elucidation of the role played by mammary epithelial cells (MECs) in fighting microbial infections to the discovery of novel diagnostic markers for monitoring udder health in dairy animals. Here, we investigated the mammary gland response following a two-step experimental infection of lactating sheep with the mastitis-associated bacterium Streptococcus uberis. The establishment of infection was confirmed both clinically and by molecular methods, including PCR and fluorescent in situ hybridization of mammary tissues. Proteomic investigation of the milk fat globule (MFG), a complex vesicle released by lactating MECs, enabled detection of enrichment of several proteins involved in inflammation, chemotaxis of immune cells, and antimicrobial defense, including cathelicidins and calprotectin (S100A8/S100A9), in infected animals, suggesting the consistent involvement of MECs in the innate immune response to pathogens. The ability of MECs to produce and release antimicrobial and immune defense proteins was then demonstrated by immunohistochemistry and confocal immunomicroscopy of cathelicidin and the calprotectin subunit S100A9 on mammary tissues. The time course of their release in milk was also assessed by Western immunoblotting along the course of the experimental infection, revealing the rapid increase of these proteins in the MFG fraction in response to the presence of bacteria. Our results support an active role of MECs in the innate immune response of the mammary gland and provide new potential for the development of novel and more sensitive tools for monitoring mastitis in dairy animals.

Development of immunodiagnostic tools for in situ investigation of Ovis aries papillomavirus 3 (OaPV3).

Cutaneous squamous cell carcinoma (cSCC) is a malignant lesion characterized by proliferation and transformation of keratinocytes in the epidermis and infiltrating derma. cSCC is reported in domestic and wild animal species, worldwide. The occurrence and development of cSCC rely on synergic multifactorial conditions, most importantly sunlight exposure and Papillomavirus (PV) infection. In sheep, the development of such lesions represents a threat both to animal welfare and milk production. Ovis aries papillomavirus 3 (OaPV3) is the main cSCC viral determinant and oncogenic properties of viral E6 and E7 proteins were preliminarily investigated. However, E6 and E7 role and mechanisms resulting in cSCC have not been fully clarified, mainly due to the lack specific immunological tools, such as antibodies for in situ detection of ovine papillomavirus. This paper reports the development of specific serological tools for the investigation of OaPV3 pathogenicity, and their preliminary use to screen 4 ovine cSSC formalin-fixed paraffin embedded tissues. Relevance of immunological tools to investigation of viral biological properties and diagnosis are also discussed.

Organic Electrochemical Transistors as Versatile Tool for Real-Time and Automatized Viral Cytopathic Effect Evaluation.

In-vitro viral studies are still fundamental for biomedical research since studying the virus kinetics on cells is crucial for the determination of the biological properties of viruses and for screening the inhibitors of infections. Moreover, testing potential viral contaminants is often mandatory for safety evaluation. Nowadays, viral cytopathic effects are mainly evaluated through end-point assays requiring dye-staining combined with optical evaluation. Recently, optical-based automatized equipment has been marketed, aimed at the real-time screening of cell-layer status and obtaining further insights, which are unavailable with end-point assays. However, these technologies present two huge limitations, namely, high costs and the possibility to study only cytopathic viruses, whose effects lead to plaque formation and layer disruption. Here, we employed poly(3,4-ethylenedioxythiophene):poly(styrene sulfonate) (Pedot:Pss) organic electrochemical transistors (OECTs) for the real-time, electrical monitoring of the infection of cytolytic viruses, i.e., encephalomyocarditis virus (EMCV), and non-cytolytic viruses, i.e., bovine coronavirus (B-CoV), on cells. OECT data on EMCV were validated using a commercially-available optical-based technology, which, however, failed in the B-CoV titration analysis, as expected. The OECTs proved to be reliable, fast, and versatile devices for viral infection monitoring, which could be scaled up at low cost, reducing the operator workload and speeding up in-vitro assays in the biomedical research field.

Proteomics and pathway analyses of the milk fat globule in sheep naturally infected by Mycoplasma agalactiae provide indications of the in vivo response of the mammary epithelium to bacterial infection.

Milk fat globules (MFGs) are vesicles released in milk as fat droplets surrounded by the endoplasmic reticulum and apical cell membranes. During formation and apocrine secretion by lactocytes, various amounts of cytoplasmic crescents remain trapped within the released vesicle, making MFGs a natural sampling mechanism of the lactating cell contents. With the aim of investigating the events occurring in the mammary epithelium during bacterial infection, the MFG proteome was characterized by two-dimensional difference gel electrophoresis (2-D DIGE), SDS-PAGE followed by shotgun liquid chromatography-tandem mass spectrometry (GeLC-MS/MS), label-free quantification by the normalized spectral abundance factor (NSAF) approach, Western blotting, and pathway analysis, using sheep naturally infected by Mycoplasma agalactiae. A number of protein classes were found to increase in MFGs upon infection, including proteins involved in inflammation and host defense, cortical cytoskeleton proteins, heat shock proteins, and proteins related to oxidative stress. Conversely, a strikingly lower abundance was observed for proteins devoted to MFG metabolism and secretion. To our knowledge, this is the first report describing proteomic changes occurring in MFGs during sheep infectious mastitis. The results presented here offer new insights into the in vivo response of mammary epithelial cells to bacterial infection and open the way to the discovery of protein biomarkers for monitoring clinical and subclinical mastitis.

Identification of conserved Mycoplasma agalactiae surface antigens by immunoproteomics.

Contagious agalactia represents one of the most relevant infectious diseases of dairy sheep, with Mycoplasma agalactiae being the primary etiological agent. The early, sensitive, and specific identification of infected animals, as well as the development of efficient prophylactic tools, remain challenging. Here, we present a comprehensive characterization of M. agalactiae antigens focusing on those shared among different isolates. Leveraging on previous proteomic data obtained on individual strains, we adopted a strategy entailing sample pooling to optimize the identification of conserved proteins that induce an immune response. The liposoluble proteins from previously characterized field isolates and the type strain PG2T were enriched by Triton X-114 fractionation, pooled, analysed by one-dimensional (1D) and two-dimensional (2D) electrophoresis, and subjected to western immunoblotting against sheep sera collected during natural infection with M. agalactiae. Immunodominant antigens were identified by Matrix-Assisted Laser Desorption-Time-Of-Flight-Mass Spectrometry (MALDI-TOF-MS). This combined immunoproteomic approach confirmed the role of several known immunogens, including P80, P48, and P40, and most variable surface proteins (Vpmas), and unveiled novel immunodominant, conserved antigens, including MAG_1000, MAG_2220, MAG_1980, phnD, MAG_4740, and MAG_2430. Genomic context, functional prediction, subcellular localization, and invariable expression of these proteins in all isolates suggest their possible involvement in bacterial pathogenicity and metabolism. Moreover, most of the identified antigens elicit a host humoral response since the early stages of infection, persisting for at least 270 days. The immunodominant, conserved antigen panel identified in this work supports the development of effective vaccines and diagnostic tools with higher sensitivity and specificity in all the natural infection stages.

Proteomic profiles and cytokeratin 13 as a potential biomarker of Ovis aries papillomavirus 3-positive and negative cutaneous squamous cell carcinomas.

Ovis aries papillomavirus 3 (OaPV3) is an epidermotropic PV reported in sheep cutaneous squamous cell carcinoma (SCC). The presence of OaPV3 DNA and its transcriptional activity in cutaneous SCC, as well as its in vitro transforming properties, suggest a viral etiology for this neoplasm. Nevertheless, the reactome associated with viral-host interaction is still unexplored. Here, we investigated and compared the proteomic profiles of OaPV3-positive SCCs, OaPV3-negative SCCs, and non-SCC samples by liquid chromatography tandem-mass spectrometry (LC-MS/MS) analysis, bioinformatics tools, and immunohistochemistry (IHC). OaPV3-positive SCCs (n = 3), OaPV3-negative SCCs (n = 3), and non-SCCs samples (n = 3) were subjected to a shotgun proteomic analysis workflow to assess protein abundance differences among the three sample classes. Proteins involved in epithelial cell differentiation, extracellular matrix organization, and apoptotic signaling showed different abundances in OaPV3-positive SCCs tissues (P ≤ 0.05) when compared to the other tissues. Cytokeratin 13 (CK 13) was among the most increased proteins in OaPV3-positive SCC and was validated by immunohistochemistry on 10 samples per class, confirming its potential as a biomarker of OaPV3 infection in SCC. Collectively, results provide a preliminary insight into the reactome associated with viral-host interaction and pave the way to the development of specific biomarkers for viral-induced sheep SCC.

The liposoluble proteome of Mycoplasma agalactiae: an insight into the minimal protein complement of a bacterial membrane.

BACKGROUND: Mycoplasmas are the simplest bacteria capable of autonomous replication. Their evolution proceeded from gram-positive bacteria, with the loss of many biosynthetic pathways and of the cell wall. In this work, the liposoluble protein complement of Mycoplasma agalactiae, a minimal bacterial pathogen causing mastitis, polyarthritis, keratoconjunctivitis, and abortion in small ruminants, was subjected to systematic characterization in order to gain insights into its membrane proteome composition. RESULTS: The selective enrichment for M. agalactiae PG2T liposoluble proteins was accomplished by means of Triton X-114 fractionation. Liposoluble proteins were subjected to 2-D PAGE-MS, leading to the identification of 40 unique proteins and to the generation of a reference 2D map of the M. agalactiae liposoluble proteome. Liposoluble proteins from the type strain PG2 and two field isolates were then compared by means of 2D DIGE, revealing reproducible differences in protein expression among isolates. An in-depth analysis was then performed by GeLC-MS/MS in order to achieve a higher coverage of the liposoluble proteome. Using this approach, a total of 194 unique proteins were identified, corresponding to 26% of all M. agalactiae PG2T genes. A gene ontology analysis and classification for localization and function was also carried out on all protein identifications. Interestingly, the 11.5% of expressed membrane proteins derived from putative horizontal gene transfer events. CONCLUSIONS: This study led to the in-depth systematic characterization of the M. agalactiae liposoluble protein component, providing useful insights into its membrane organization.

Proteomic datasets of uninfected and Staphylococcus aureus-infected goat milk.

We present a proteomic dataset generated from half-udder Alpine goat milk. The milk samples belonged to 3 groups: i) mid-lactation, low somatic cell count, uninfected milk (MLU, n=3); ii) late lactation, high somatic cell count, uninfected milk (LHU, n=3); and late lactation, high somatic cell count, Staphylococcus aureus subclinically infected milk (LHS, n=3). The detailed description of results is reported in the research article entitled "Impact of Staphylococcus aureus infection on the late lactation goat milk proteome: new perspectives for monitoring and understanding mastitis in dairy goats". After milk defatting, high speed centrifugation and trypsin digestion of milk with the FASP protocol, peptide mixtures were analyzed by LC-MS/MS on a Q-Exactive. Peptide identification was carried out using Sequest-HT in Proteome Discoverer. Then, the Normalized Abundance Spectrum Factor (NSAF) value was calculated by label free quantitation using the spectral counting approach, and Gene Ontology (GO) annotation by Uniprot was carried out by reporting biological process, molecular function and cellular component. The MS data have been deposited to the ProteomeXchange via the PRIDE with the dataset identifier PXD017243.

Impact of Staphylococcus aureus infection on the late lactation goat milk proteome: New perspectives for monitoring and understanding mastitis in dairy goats.

The milk somatic cell count (SCC) is a standard parameter for monitoring intramammary infections (IMI) in dairy ruminants. In goats, however, the physiological increase in SCC occurring in late lactation heavily compromises its reliability. To identify and understand milk protein changes specifically related to IMI, we carried out a shotgun proteomics study comparing high SCC late lactation milk from goats with subclinical Staphylococcus aureus IMI and from healthy goats to low SCC mid-lactation milk from healthy goats. As a result, we detected 52 and 19 differential proteins (DPs) in S. aureus-infected and uninfected late lactation milk, respectively. Unexpectedly, one of the proteins higher in uninfected milk was serum amyloid A. On the other hand, 38 DPs were increased only in S. aureus-infected milk and included haptoglobin and numerous cytoskeletal proteins. Based on STRING analysis, the DPs unique to S. aureus infected milk were mainly involved in defense response, cytoskeleton organization, cell-to-cell, and cell-to-matrix interactions. Being tightly and specifically related to infectious/inflammatory processes, these proteins may hold promise as more reliable markers of IMI than SCC in late lactation goats. SIGNIFICANCE: The biological relevance of our results lies in the increased understanding of the changes specifically related to bacterial infection of the goat udder in late lactation. The DPs present only in S. aureus infected milk may find application as markers for improving the specificity of subclinical mastitis monitoring and detection in dairy goats in late lactation, when other widespread tools such as the SCC lose diagnostic value.

Molecular epidemiology of Anaplasma spp. related to A. phagocytophilum in Mediterranean small ruminants.

The genus Anaplasma currently comprises 6 bacterial species mostly pathogenic to animals and/or human, including the zoonotic species Anaplasma phagocytophilum, the causative agent of tick-borne fever (TBF) of ruminants, and of granulocytic anaplasmosis of horses, dogs and human. Recently, novel potentially non-pathogenic strains related to A. phagocytophilum have been identified in Japan, China, and Tunisia. This paper reports the identification, molecular typing, and evolutionary history of novel Anaplasma strains (A. phagocytophilum-like 1 and 2), related to but distinct from A. phagocytophilum in Mediterranean area of Europe and Africa. PCR-RFLP and phylogenetic analyses based on 16S rRNA provided evidence for the circulation of A. phagocytophilum-like 1 strains in Europe. Phylogeny based on groEL gene showed the inclusion of Sardinian and Tunisian A. phagocytophilum-like 1 strains in a unique clade distinct from, but related to that of Japanese strains. Results suggest that genetic diversity within the genus Anaplasma is much greater than expected and provide information useful for the development of specific and effective diagnostic and prophylactic tools.

Detection and Characterization of an Avipoxvirus in a Common Buzzard ( Buteo buteo) in Italy Using a Multiple Gene Approach.

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.