Osteogenic differentiation of adipose-derived canine mesenchymal stem cells seeded in porous calcium-phosphate scaffolds.

Introduction: Engineered bone graft substitutes are a promising alternative and supplement to autologous bone grafts as treatments for bone healing impairment. Advances in human medicine extend an invitation to pursue these biomimetic strategies in animal patients, substantiated by the theory that specialized scaffolds, multipotent cells, and biological cues may be combined into a bioactive implant intended for the enhancement of tissue regeneration. Methods: This proof-of-concept study was designed to evaluate and validate the feasibility of beta-tricalcium phosphate foam scaffolds seeded with canine mesenchymal stem cells derived from adipose tissue. Cell-inoculated samples and sham controls were cultured statically for 72 hours in complete growth medium to evaluate seeding capacity, while a subset of loaded scaffolds was further induced with osteogenic culture medium for 21 days. Produced implants were characterized and validated with a combination of immunofluorescence and reflection confocal microscopy, scanning electron microscopy, and polymerase chain reaction to confirm osteogenic differentiation in tridimensional-induced samples. Results: After 72 hours of culture, all inoculated scaffolds presented widespread yet heterogeneous surface seeding, distinctively congregating stem cells around pore openings. Furthermore, at 21 days of osteogenic culture conditions, robust osteoblastic differentiation of the seeded cells was confirmed by the change of cell morphology and evident deposition of extra-cellular matrix, accompanied by mineralization and scaffold remodeling; furthermore, all induced cell-loaded implants lost specific stemness immunophenotype expression and simultaneously upregulated genomic expression of osteogenic genes Osterix and Ostecalcin. Conclusions: ß-TCP bio-ceramic foam scaffolds proved to be suitable carriers and hosts of canine adipose-derived MSCs, promoting not only surface attachment and proliferation, but also demonstrating strong in-vitro osteogenic potential. Although this research provides satisfactory in-vitro validation for the conceptualization and feasibility of a canine bio-active bone implant, further testing such as patient safety, large-scale reproducibility, and quality assessment are needed for regulatory compliance in future commercial clinical applications.

3D printing with star-shaped strands: A new approach to enhance in vivo bone regeneration.

Concave surfaces have shown to promote bone regeneration in vivo. However, bone scaffolds obtained by direct ink writing, one of the most promising approaches for the fabrication of personalized bone grafts, consist mostly of convex surfaces, since they are obtained by microextrusion of cylindrical strands. By modifying the geometry of the nozzle, it is possible to print 3D structures composed of non-cylindrical strands and favor the presence of concave surfaces. In this work, we compare the in vivo performance of 3D-printed calcium phosphate scaffolds with either conventional cylindrical strands or star-shaped strands, in a rabbit femoral condyle model. Monocortical defects, drilled in contralateral positions, are randomly grafted with the two scaffold configurations, with identical composition. The samples are explanted eight weeks post-surgery and assessed by µ-CT and resin-embedded histological observations. The results reveal that the scaffolds containing star-shaped strands have better osteoconductive properties, guiding the newly formed bone faster towards the core of the scaffolds, and enhance bone regeneration, although the increase is not statistically significant (p > 0.05). This new approach represents a turning point towards the optimization of pore shape in 3D-printed bone grafts, further boosting the possibilities that direct ink writing technology offers for patient-specific applications.

On-Growth and In-Growth Osseointegration Enhancement in PM Porous Ti-Scaffolds by Two Different Bioactivation Strategies: Alkali Thermochemical Treatment and RGD Peptide Coating.

A lack of primary stability and osteointegration in metallic implants may result in implant loosening and failure. Adding porosity to metallic implants reduces the stress shielding effect and improves implant performance, allowing the surrounding bone tissue to grow into the scaffold. However, a bioactive surface is needed to stimulate implant osteointegration and improve mechanical stability. In this study, porous titanium implants were produced via powder sintering to create different porous diameters and open interconnectivity. Two strategies were used to generate a bioactive surface on the metallic foams: (1) an inorganic alkali thermochemical treatment, (2) grafting a cell adhesive tripeptide (RGD). RGD peptides exhibit an affinity for integrins expressed by osteoblasts, and have been reported to improve osteoblast adhesion, whereas the thermochemical treatment is known to improve titanium implant osseointegration upon implantation. Bioactivated scaffolds and control samples were implanted into the tibiae of rabbits to analyze the effect of these two strategies in vivo regarding bone tissue regeneration through interconnected porosity. Histomorphometric evaluation was performed at 4 and 12 weeks after implantation. Bone-to-implant contact (BIC) and bone in-growth and on-growth were evaluated in different regions of interest (ROIs) inside and outside the implant. The results of this study show that after a long-term postoperative period, the RGD-coated samples presented higher quantification values of quantified newly formed bone tissue in the implant's outer area. However, the total analyzed bone in-growth was observed to be slightly greater in the scaffolds treated with alkali thermochemical treatment. These results suggest that both strategies contribute to enhancing porous metallic implant stability and osteointegration, and a combination of both strategies might be worth pursuing.

Hydrothermal processing of 3D-printed calcium phosphate scaffolds enhances bone formation in vivo: a comparison with biomimetic treatment.

Hydrothermal (H) processes accelerate the hydrolysis reaction of α-tricalcium phosphate (α-TCP) compared to the long-established biomimetic (B) treatments. They are of special interest for patient-specific 3D-printed bone graft substitutes, where the manufacturing time represents a critical constraint. Altering the reaction conditions has implications for the physicochemical properties of the reaction product. However, the impact of the changes produced by the hydrothermal reaction on the in vivo performance was hitherto unknown. The present study compares the bone regeneration potential of 3D-printed α-TCP scaffolds hardened using these two treatments in rabbit condyle monocortical defects. Although both consolidation processes resulted in biocompatible scaffolds with osseointegrative and osteoconductive properties, the amount of newly formed bone increased by one third in the hydrothermal vs the biomimetic samples. B and H scaffolds consisted mostly of high specific surface area calcium-deficient hydroxyapatite (38 and 27 m2 g-1, respectively), with H samples containing also 10 wt.% ß-tricalcium phosphate (ß-TCP). The shrinkage produced during the consolidation process was shown to be very small in both cases, below 3%, and smaller for H than for B samples. The differences in the in vivo performance were mainly attributed to the distinct crystallisation nanostructures, which proved to have a major impact on permeability and protein adsorption capacity, using BSA as a model protein, with B samples being highly impermeable. Given the crucial role that soluble proteins play in osteogenesis, this is proposed to be a relevant factor behind the distinct in vivo performances observed for the two materials. STATEMENT OF SIGNIFICANCE: The possibility to accelerate the consolidation of self-setting calcium phosphate inks through hydrothermal treatments has aroused great interest due to the associated advantages for the development of 3D-printed personalised bone scaffolds. Understanding the implications of this approach on the in vivo performance of the scaffolds is of paramount importance. This study compares, for the first time, this treatment to the long-established biomimetic setting strategy in terms of osteogenic potential in vivo in a rabbit model, and relates the results obtained to the physicochemical properties of the 3D-printed scaffolds (composition, crystallinity, nanostructure, nanoporosity) and their interaction with soluble proteins.

Use of three-dimensionally printed ß-tricalcium phosphate synthetic bone graft combined with recombinant human bone morphogenic protein-2 to treat a severe radial atrophic nonunion in a Yorkshire terrier.

OBJECTIVE: To describe a novel surgical approach to treat a critical-sized bone defect due to severe, radial atrophic nonunion in a miniature dog. STUDY DESIGN: Case report ANIMAL: A 1-year-old Yorkshire terrier with a critical-sized left radial defect after failed internal fixation of a transverse radial fracture. METHODS: Computed tomographic (CT) images of the radius were imported for three-dimensional (3D) printing of a custom-designed synthetic 3D-printed ß-tricalcium phosphate (ß-TCP) scaffold. The radius was exposed, and the ß-TCP scaffold was press-fitted in the bone gap underneath the plate. Recombinant human bone morphogenic protein-2 (RhBMP-2) collagen sponges were squeezed to soak the scaffold with growth factor and then placed on both sides of the synthetic graft. Two additional cortical screws were also placed prior to routine closure of the surgical site. RESULTS: Radiographic examination was consistent with complete healing of the radius defect 4 months after surgery. The bone plate was removed 10 months after surgery. According to CT examination 18 months after surgery, there was no evidence of the synthetic graft; instead, complete corticalization of the affected area was noted. Complete functional recovery was observed until the last clinical follow-up 36 months postoperatively. CONCLUSION: Screw fixation and use of a 3D-printed ceramic scaffold augmented with rhBMP-2 resulted in excellent bone regeneration of the nonunion and full recovery of a miniature breed dog. CLINICAL SIGNIFICANCE: The therapeutic approach used in this dog could be considered as an option for treatment of large-bone defects in veterinary orthopedics, especially for defects affecting the distal radius of miniature dogs.

Osseointegration around dental implants biofunctionalized with TGFß-1 inhibitor peptides: an in vivo study in beagle dogs.

The aim of this study was to evaluate the effect of biofunctionalization with two TGF-ß1 inhibitor peptides, P17 and P144, on osseointegration of CP-Ti dental implants. A total of 36 implants (VEGA, Klockner®) with 3.5 × 8 mm internal connection were used in this study, divided in three groups: (1) control group (n = 12), (2) implants which surfaces were biofunctionalized with P17 peptide inhibitor (n = 12), (3) implants with surfaces biofunctionalized by P144 peptide (n = 12). Three implants, one from each group, were inserted in both hemimandibles of 6 beagle dogs, 2 months after tooth extraction. Two animals were sacrificed at 2, 4 and 8 weeks post implant insertion, respectively. The samples were analyzed by Backscattering Scanning Electron Microscopy (BS-SEM) and histological analysis. Histomorphometric analysis of bone to implant contact (BIC), peri-implant bone fraction (BF) and interthread bone (IB) were carried out. Bone formation around implants measured by quantitative analysis, BS-SEM, was significantly higher in the P17-biofunctionalized implants, 4 and 8 weeks after the implantation. Histomorphometric analysis of BIC, BF and IB showed higher values in the P17-biofunctionalized group at initial stages of healing (2 weeks) and early osseointegration both at 4 and 8 weeks. For P144 biofunctionalized implants, the histomorphometric values obtained are also higher than control group. Accordingly, better results in the experimental groups were proven both by the quantitative and the qualitative analysis. Surface biofunctionalization with TGF-ß1 inhibitor peptides, P17 and P144, resulted in better quantitative and qualitative parameters relative to implant osseointegration.

Impact of Biomimicry in the Design of Osteoinductive Bone Substitutes: Nanoscale Matters.

Bone apatite consists of carbonated calcium-deficient hydroxyapatite (CDHA) nanocrystals. Biomimetic routes allow fabricating synthetic bone grafts that mimic biological apatite. In this work, we explored the role of two distinctive features of biomimetic apatites, namely, nanocrystal morphology (plate vs needle-like crystals) and carbonate content, on the bone regeneration potential of CDHA scaffolds in an in vivo canine model. Both ectopic bone formation and scaffold degradation were drastically affected by the nanocrystal morphology after intramuscular implantation. Fine-CDHA foams with needle-like nanocrystals, comparable in size to bone mineral, showed a markedly higher osteoinductive potential and a superior degradation than chemically identical coarse-CDHA foams with larger plate-shaped crystals. These findings correlated well with the superior bone-healing capacity showed by the fine-CDHA scaffolds when implanted intraosseously. Moreover, carbonate doping of CDHA, which resulted in small plate-shaped nanocrystals, accelerated both the intrinsic osteoinduction and the bone healing capacity, and significantly increased the cell-mediated resorption. These results suggest that tuning the chemical composition and the nanostructural features may allow the material to enter the physiological bone remodeling cycle, promoting a tight synchronization between scaffold degradation and bone formation.

Osteogenesis by foamed and 3D-printed nanostructured calcium phosphate scaffolds: Effect of pore architecture.

There is an urgent need of synthetic bone grafts with enhanced osteogenic capacity. This can be achieved by combining biomaterials with exogenous growth factors, which however can have numerous undesired side effects, but also by tuning the intrinsic biomaterial properties. In a previous study, we showed the synergistic effect of nanostructure and pore architecture of biomimetic calcium deficient hydroxyapatite (CDHA) scaffolds in enhancing osteoinduction, i.e. fostering the differentiation of mesenchymal stem cells to bone forming cells. This was demonstrated by assessing bone formation after implanting the scaffolds intramuscularly. The present study goes one step forward, since it analyzes the effect of the geometrical features of the same CDHA scaffolds, obtained either by 3D-printing or by foaming, on the osteogenic potential and resorption behaviour in a bony environment. After 6 and 12 weeks of intraosseous implantation, both bone formation and material degradation had been drastically affected by the macropore architecture of the scaffolds. Whereas nanostructured CDHA was shown to be highly osteoconductive both in the robocast and foamed scaffolds, a superior osteogenic capacity was observed in the foamed scaffolds, which was associated with their higher intrinsic osteoinductive potential. Moreover, they showed a significantly higher cell-mediated degradation than the robocast constructs, with a simultaneous and progressive replacement of the scaffold by new bone. In conclusion, these results demonstrate that the control of macropore architecture is a crucial parameter in the design of synthetic bone grafts, which allows fostering both material degradation and new bone formation. Statement of Significance 3D-printing technologies open new perspectives for the design of patient-specific bone grafts, since they allow customizing the external shape together with the internal architecture of implants. In this respect, it is important to design the appropriate pore geometry to maximize the bone healing capacity of these implants. The present study analyses the effect of pore architecture of nanostructured hydroxyapatite scaffolds, obtained either by 3D-printing or foaming, on the osteogenic potential and scaffold resorption in an in vivo model. While nanostructured hydroxyapatite showed excellent osteoconductive properties irrespective of pore geometry, we demonstrated that the spherical, concave macropores of foamed scaffolds significantly promoted both material resorption and bone regeneration compared to the 3D-printed scaffolds with orthogonal-patterned struts and therefore prismatic, convex macropores.

Two Different Strategies to Enhance Osseointegration in Porous Titanium: Inorganic Thermo-Chemical Treatment Versus Organic Coating by Peptide Adsorption.

In this study, highly-interconnected porous titanium implants were produced by powder sintering with different porous diameters and open interconnectivity. The actual foams were produced using high cost technologies: Chemical Vapor Deposition (CVD), Physical Vapor Deposition (PVD), and spark plasma sintering, and the porosity and/or interconnection was not optimized. The aim was to generate a bioactive surface on foams using two different strategies, based on inorganic thermo-chemical treatment and organic coating by peptide adsorption, to enhance osseointegration. Porosity was produced using NaCl as a space holder and polyethyleneglicol as a binder phase. Static and fatigue tests were performed in order to determine mechanical behaviors. Surface bioactivation was performed using a thermo-chemical treatment or by chemical adsorption with peptides. Osteoblast-like cells were cultured and cytotoxicity was measured. Bioactivated scaffolds and a control were implanted in the tibiae of rabbits. Histomorphometric evaluation was performed at 4 weeks after implantation. Interconnected porosity was 53% with an average diameter of 210 µm and an elastic modulus of around 1 GPa with good mechanical properties. The samples presented cell survival values close to 100% of viability. Newly formed bone was observed inside macropores, through interconnected porosity, and on the implant surface. Successful bone colonization of inner structure (40%) suggested good osteoconductive capability of the implant. Bioactivated foams showed better results than non-treated ones, suggesting both bioactivation strategies induce osteointegration capability.

Osteoinduction by Foamed and 3D-Printed Calcium Phosphate Scaffolds: Effect of Nanostructure and Pore Architecture.

Some biomaterials are osteoinductive, that is, they are able to trigger the osteogenic process by inducing the differentiation of mesenchymal stem cells to the osteogenic lineage. Although the underlying mechanism is still unclear, microporosity and specific surface area (SSA) have been identified as critical factors in material-associated osteoinduction. However, only sintered ceramics, which have a limited range of porosities and SSA, have been analyzed so far. In this work, we were able to extend these ranges to the nanoscale, through the foaming and 3D-printing of biomimetic calcium phosphates, thereby obtaining scaffolds with controlled micro- and nanoporosity and with tailored macropore architectures. Calcium-deficient hydroxyapatite (CDHA) scaffolds were evaluated after 6 and 12 weeks in an ectopic-implantation canine model and compared with two sintered ceramics, biphasic calcium phosphate and ß-tricalcium phosphate. Only foams with spherical, concave macropores and not 3D-printed scaffolds with convex, prismatic macropores induced significant ectopic bone formation. Among them, biomimetic nanostructured CDHA produced the highest incidence of ectopic bone and accelerated bone formation when compared with conventional microstructured sintered calcium phosphates with the same macropore architecture. Moreover, they exhibited different bone formation patterns; in CDHA foams, the new ectopic bone progressively replaced the scaffold, whereas in sintered biphasic calcium phosphate scaffolds, bone was deposited on the surface of the material, progressively filling the pore space. In conclusion, this study demonstrates that the high reactivity of nanostructured biomimetic CDHA combined with a spherical, concave macroporosity allows the pushing of the osteoinduction potential beyond the limits of microstructured calcium phosphate ceramics.

In Vivo Osteogenic Potential of Biomimetic Hydroxyapatite/Collagen Microspheres: Comparison with Injectable Cement Pastes.

The osteogenic capacity of biomimetic calcium deficient hydroxyapatite microspheres with and without collagen obtained by emulsification of a calcium phosphate cement paste has been evaluated in an in vivo model, and compared with an injectable calcium phosphate cement with the same composition. The materials were implanted into a 5 mm defect in the femur condyle of rabbits, and bone formation was assessed after 1 and 3 months. The histological analysis revealed that the cements presented cellular activity only in the margins of the material, whereas each one of the individual microspheres was covered with osteogenic cells. Consequently, bone ingrowth was enhanced by the microspheres, with a tenfold increase compared to the cement, which was associated to the higher accessibility for the cells provided by the macroporous network between the microspheres, and the larger surface area available for osteoconduction. No significant differences were found in terms of bone formation associated with the presence of collagen in the materials, although a more extensive erosion of the collagen-containing microspheres was observed.

Ultrasound guidance for the performance of sciatic and saphenous nerve blocks in dogs.

The aim of this study was to investigate the use of ultrasound (US) guidance to perform sciatic and saphenous nerve blocks in dogs. Five dogs were sedated with medetomidine and butorphanol. A high-resolution US transducer was used to locate the nerves, guide placement of the needle and visualise the perineural injection of lidocaine 2%. Electrostimulation was used to confirm correct placement prior to the sciatic block. Nerve functions were evaluated over a 3 h period following administration of atipamezole. Successful identification of the nerves and the quality of the blocks were recorded. Location of the nerves, complete sensory block of the saphenous nerve, and partial to complete sensory and motor blocks of the sciatic nerve were achieved in all dogs. The resultant US guidance is potentially valuable for blocking the sciatic and saphenous nerves in dogs, although further work will be required to ensure a complete block of the sciatic nerve.

The stepped hybrid plate for carpal panarthrodesis - Part II: a multicentre study of 52 arthrodeses.

Fifty-two carpal panarthrodeses (CP) were carried out in 44 dogs (eight bilateral), in a multicentre study using a single (n = 47) or double (n = 5) stepped hybrid CP plate. Of these 44 cases, 39 were between 20-55 kg in bodyweight , 26 were males , and the carpometacarpal was the most common joint involved. Falling and other impact trauma were the most common aetiology. Pain of unknown origin, carpal luxation, chronic accessory carpal bone fracture, distal comminuted intra-articular radial fracture, bone tumour, degenerative joint disease, canine erosive idiopathic polyarthritis, avascular necrosis of the radial carpal bone and fractures of several metacarpal bones were some of the pathologies reported. Fracture of the third metacarpal bone during screw insertion was the only intrasurgical complication. Malpositioning of the plate or screws and over-tightened screws were technical errors observed in seven of the procedures. The radial carpal bone was not fixed with a screw in two cases due to bone deformity. Concurrent plate breakage and bending in the same patient operated on bilaterally was observed during the follow-up period, which represented a major complication rate of 3.8% for all procedures. Minor complications were: low grade infection, lick granuloma, digit hyperextension, screw loosening or failure, incomplete fusion of some joints and a fracture of the third metacarpal bone at the distal screw hole of the plate; which represented a rate of 44.2% on all procedures. Complete carpal healing was observed radiographically in 94.2% of all procedures. Limb function was excellent to good, and all of the owners, except for one, were satisfied with the procedure.

Experimental study of bone lengthening in dogs by means of backscattered scanning electron microscopy.

OBJECTIVE: To describe the morphology of calcified tissues involved in distraction osteogenesis (DO) by means of backscattered scanning electron microscopy (BS-SEM). STUDY DESIGN: Experimental study. ANIMALS: Adult female Beagle dogs (n=12). METHODS: Non-simultaneous and bilateral transverse mid-diaphyseal osteotomies performed in tibiae were stabilized and distracted by a Type Ia external skeletal fixation device. After a latency period of 5 days, distraction was applied at a rate of 0.5 mm every 12 hours for 10 days. Then, the external fixator was maintained in a static mode during the consolidation period until bone healing or euthanasia at 1, 2, 3, 4, 6, 8, 10, 12, 14, and 18 weeks after operations, whichever came first. Distracted regions were isolated and their structure was examined by BS-SEM. RESULTS: Calcified chondroid tissue was prominent during distraction and calcified cartilaginous tissue during consolidation; both tissues were successively replaced by woven, lamellar, and osteonal bone. CONCLUSIONS: In osteotomized tibia, chondroid tissue is the main component of the mineralization front during distraction, calcified cartilaginous tissue during consolidation, and then both tissues are replaced by woven, lamellar, and osteonal bone. The ossification mechanism of distraction callus is transchondroidal. CLINICAL RELEVANCE: BS-SEM is an effective technique for studying progression of bone healing during DO. The presence of chondroid tissue during DO explains why callus mineralization occurs more rapidly during distraction than during static stabilization.

Cervical and coelomic radiologic features of the loggerhead sea turtle, Caretta caretta.

Many investigators have undertaken radiologic studies in chelonians. However, descriptive papers focusing on the radiographic anatomy are limited to only a few species. The purpose of this article is to provide the normal cervical and coelomic radiographic appearance of the loggerhead sea turtle (Caretta caretta), in the dorsoventral view, and to indicate useful landmarks to identify internal anatomic structures. Dorsoventral radiographs were taken of the neck and body of 30 loggerhead sea turtles by means of analog and digital radiography. At various points, distortion or superimposition of images due to the natural curvature of the shell hindered the accuracy of interpretation. The pectoral and pelvic girdles were easily recognized. Important external landmarks included the vertebral and lateral scutes, and important internal landmarks included the bronchi, coracoid bones, the caudal border of the pulmonary fields, and the acetabulum.

Comparison between meloxicam and transdermally administered fentanyl for treatment of postoperative pain in dogs undergoing osteotomy of the tibia and fibula and placement of a uniplanar external distraction device.

OBJECTIVE: To compare the efficacy of meloxicam administered perioperatively with transdermal administration of fentanyl via a patch placed preoperatively in dogs undergoing orthopedic surgery. DESIGN: Prospective study. ANIMALS: 16 dogs. PROCEDURE: Unilateral or bilateral osteotomy of the tibia and fibula was surgically performed, and a uniplanar external distraction device was placed in each limb. Postoperative pain and lameness were assessed 24, 48, and 72 hours after administration of the first of 3 doses of meloxicam (0.2 mg/kg [0.09 mg/lb], IV, given preoperatively, followed by 0.1 mg/kg [0.045 mg/lb], IV, after 24 hours, and 0.1 mg/kg, PO, after 48 hours) or preoperative placement of a transdermal fentanyl patch (50 microg/h) left in place for 72 hours. RESULTS: No significant differences in total pain scores were detected between groups. Mean +/- SD lameness scores assessed at 24 and 72 hours were lower in dogs in the meloxicam group than dogs in the fentanyl group. Lameness scores decreased with time in a similar manner in both treatment groups. CONCLUSIONS AND CLINICAL RELEVANCE: Perioperative administration of meloxicam or preoperative placement of a transdermal fentanyl patch provided effective and similar postoperative analgesia in dogs undergoing orthopedic surgery. However, because of its anti-inflammatory effects, treatment with meloxicam reduced the degree of lameness and resulted in rapid functional recovery of the limb.

Foot infections associated with Arcanobacterium pyogenes in free-living fallow deer (Dama dama).

We describe foot infection associated with Arcanobacterium pyogenes in three adult male free-living fallow deer (Dama dama) from Sueve Regional Hunting Reserve (Principality of Asturias, Spain). Affected fallow deer were culled in November 1997 and 1998 during the hunting season. Necropsy, radiography, and microbiologic analysis were carried out for each animal. Unilateral swelling of one extremity at the coronary band was observed in all three cases. Areas of bone loss, severe periosteal reaction, and soft tissue swelling were seen on radiography. Lead fragments were observed in one fallow deer. Seven bacterial species were isolated, but only Arcanobacterium pyogenes was routinely found. Weather conditions in the area (mild temperatures and high humidity), the land (alternating pasture land and rock), the animal population density (both fallow deer and domestic herds of cows, horses, sheep, and goats, live side by side in the same areas), and hunting activities could be related to the frequency of these infections.