Management of Dental Disease in Aardvarks (Orycteropus afer) and Potential Use of Cone-Beam Computed Tomography.

Oral disease involving teeth is a common cause of morbidity in aardvarks (Orycteropus afer) under managed care. Cases can be challenging due to the species' unique skull and dental anatomy and limited veterinary literature. A retrospective evaluation was performed on dental examinations in nine aardvarks housed at a single zoological institution in the United States between 1995 and 2021. The prevalence of dental disease in this population was 88%, with most cases categorized as mild (4/8). Clinical signs were only seen in three cases. Facial swelling prior to surgery was the most common clinical sign (3/8). Dental pathology was more common in the mandibular teeth (27/38) compared to the maxillary teeth (11/38). Dental abnormalities found upon intraoral examination included the presence of dental points (7/8), crown elongation (3/8), purulent material within the oral cavity (4/8), loose teeth (2/8), periodontal pockets (2/8), and oronasal fistula (1/8). Three patients required dental extractions with a lateral buccostomy approach. Diagnostic imaging was performed in most cases (7/8), with two cases undergoing cone-beam computed tomography (CBCT) to characterize dental pathology that was difficult to fully evaluate with standard radiography. Tomographic findings are described in both cases. CBCT was found to be a helpful tool for diagnosing and characterizing dental disease in aardvarks.

AUSTWICKIA CHELONAE IN A WILD GOPHER TORTOISE (GOPHERUS POLYPHEMUS) AND EVIDENCE OF POSITIVE SELECTION ON THE DIPHTHERIA-LIKE TOXIN GENE.

Austwickia (Dermatophilus) chelonae is a filamentous, Gram-positive Actinobacteria in the Dermatophilaceae family. It has caused fatal granulomatous disease in diverse captive reptile species on three continents, but its presence in wild or free-ranging populations was unknown. An adult female gopher tortoise (Gopherus polyphemus) was presented euhydrated, but cachectic and infested with ticks, with two firm, encapsulated masses over the cranioventral neck and right stifle. The tortoise had moderate nonregenerative anemia and evidence of inflammation; plasma biochemistry data was within normal limits. Fine needle aspirate of the neck lesion revealed abundant necrosis and aggregates of cocci. Computed tomography delineated the masses and revealed an additional mass adjacent to the left zygomatic bone. After surgical excision, histology identified chronic granulomas with intralesional filamentous bacteria. Pan-bacterial 16S rRNA PCR and sequencing of the masses identified A. chelonae. Despite treatment with oxytetracycline and ceftazidime, the tortoise deteriorated and was euthanatized. An esophageal lesion consistent with A. chelonae was seen on postmortem examination, although it was determined that the tortoise ultimately succumbed to fungal pneumonia caused by Metarhizium robertsii, an entomopathogenic biotoxin sprayed as insect control. This case reveals A. chelonae is present in free-ranging chelonians in North America. This organism produces a toxin gene similar to diphtheria toxin, one of the most potent known biotoxins, which has not been previously identified outside the genus Corynebacterium. Novel PCR primers were designed for the toxin and rpoB genes, which were amplified and sequenced from two cases and compared with two available genomes. Selection analysis revealed that the toxin gene is under positive selection, which implies it interacts significantly with the immune system, making it a good candidate for immunodiagnostic test development.

THE USE OF INTRADERMAL ALLERGY TESTING FOR ALLERGIC DERMATITIS IN PTEROPID BATS AND TREATMENT WITH ALLERGEN SPECIFIC IMMUNOTHERAPY: A CASE SERIES.

This case series describes the diagnosis of allergic dermatitis and management with allergen-specific immunotherapy (ASIT) based on intradermal allergy testing (IDAT) and adjunctive medical therapy in six pteropid bats; five large flying foxes (Pteropus vampyrus); and one variable flying fox (Pteropus hypomelanus). The cases ranged from 2 to 15 yr of age at the time of presentation. Clinical signs varied between individuals and included moist ulcerative cutaneous lesions in nonhaired skin, blepharoconjunctivitis, alopecia, and pruritus. All bats underwent IDAT under general anesthesia, and reactive allergens included a mixture of grasses, trees, weeds, and biting insects. Three of the six cases (50%) had reformulation of the ASIT before control of clinical signs was seen, and two bats were treated with the addition of oclacitinib (Apoquel). Severe adverse effects were not identified; however, one bat had self-limiting swelling at the immunotherapy injection site. All six cases showed improvement of clinical signs and perceived comfort level, including in subsequent allergy seasons.

Clinical Manifestation and Molecular Characterization of a Novel Member of the Nannizziopsiaceae in a Pulmonary Granuloma From a Galapagos Tortoise (Chelonoidis nigra).

Nannizziopsiaceae is a family of fungal organisms within the order Onygenales containing two genera of important reptile pathogens, Nannizziopsis and Paranannizziopsis. A captive Galapagos tortoise (Chelonoidis nigra) from Boca Raton, Florida, United States, was presented for a clinical history of chronic progressive lethargy and inappetence. At initial presentation, the tortoise had a moderate non-regenerative anemia, leukocytosis, whip-like heterophil projections, erythrocyte fragmentation, and fibrin strands, with the latter two raising concern for disseminated intravascular coagulation. A single large encapsulated pulmonary granuloma was identified through imaging, including plain film radiography and bronchoscopy. Direct intralesional samples were obtained from transcarapacial celioscopy for fungal culture, cytology, histopathology, and polymerase chain reaction. Amplification and sequencing of the ITS2 region of the rRNA genes with Bayesian and maximum likelihood analyses placed the fungus in the family Nannizziopsiaceae within the order Onygenales, representing a novel fungal species.

Enhancement of BMP-2 induced bone regeneration by SDF-1α mediated stem cell recruitment.

Treatment of critical size bone defects is challenging. Recent studies showed that the cytokine stromal cell-derived factor 1 alpha (SDF-1α) has potential to improve the bone regenerative effect of low bone morphogenetic protein 2 (BMP-2) concentrations. The goal of this study was to demonstrate the combined effect of SDF-1α and BMP-2 on bone regeneration and stem cell recruitment using a critical size femoral bone defect model. A total of 72 mice were randomized to six groups. External fixators were implanted onto the right femur of each mouse and 3 mm defects were created. Depending on the group affiliation, adenovirally activated fat tissue grafts expressing SDF-1α or/and BMP-2 were implanted at the defect site. One day after operation, 1×106 murine mesenchymal stromal cells (MSCs), lentivirally transduced to express the gene enhanced green fluorescent protein (eGFP), firefly luciferase, and CXCR4 were injected systemically in selected groups. Migration of the injected MSCs was observed by bioluminescence imaging on days 0, 2, 4, 6, 8, 10, 12, 14, 21, 28, and 42. After 6 weeks, animals were euthanized and 80 µm CT-scans were performed. For histological investigations, hematoxylin and eosin-, tartrate-resistant acid phosphatase-, alkaline phosphatase-, and anti-eGFP-stained sections were prepared. BMP-2 and SDF-1α combined at the defect site increased bone volume (BV) (2.72 mm³; 95% CI 1.95-3.49 mm³) compared with the negative control group (1.80 mm³; 95% CI 1.56-2.04 mm³; p<0.05). In addition, histological analysis confirmed a higher degree of bone healing in the BMP-2 and SDF-1α combined group compared with the negative control group. Bioluminescence imaging demonstrated higher numbers of migrated MSCs toward the defect site in the presence of both BMP-2 and SDF-1α at the defect site. Furthermore, eGFP-labeled migrated MSCs were found in all defect areas, when cells were injected. The ratio of osteoblasts to osteoclasts, assessed by immunohistological staining, was higher and thus showed a trend toward more bone formation for the combined use of BMP-2 and SDF-1α compared with all other groups. This study demonstrated that SDF-1α enhanced BMP-2 mediated bone healing in a critical size segmental bone defect model. Notably, both proteins alone also provided a cumulative effect on MSC attraction toward the site of injury.

Stem cell attraction via SDF-1α expressing fat tissue grafts.

Mesenchymal stromal cell (MSCs) are key cellular components for site-specific tissue regeneration. The chemokine stromal derived factor 1 alpha (SDF-1α) is known to attract stem cells via the C-X-C chemokine receptor-4 (CXCR4) receptor. The aim of the study was to develop a model for stem cell attraction using SDF-1α overexpressing fat tissue grafts. Murine MSCs were lentiviral transduced to express the genes for enhanced green fluorescent protein, firefly luciferace, and human CXCR4 (hCXCR4). Murine fat tissue was adenoviral transduced to express SDF-1α and red fluorescent protein transgenes. MSCs were cultured on transwells with SDF-1α containing supernatants from transduced fat tissue. The numbers of migrated MSCs in four groups (with hCXCR4 positive (+) or hCXCR4 negative (-) MSCs with or without SDF-1α containing supernatant) were investigated. After 36 h of culture, 9025 ± 925 cells migrated through the membrane of the transwells in group 1 (CXCR4+/SDF-1α+), 4817 ± 940 cells in group 2 (CXCR4-/SDF-1α+), 2050 ± 766 cells in group 3 (CXCR4+/SDF-1α-), and 2108 ± 426 cells in group 4 (CXCR4-/SDF-1α-). Both, the presence of SDF-1α and the expression of hCXCR4 significantly increased the migration rates (p < 0.0001). MSCs overexpressing the CXCR4 receptor by lentiviral transduction are highly attracted by medium from SDF-1α expressing fat tissue in vitro. Thus, SDF-1α activated tissue grafts may be a strategy to enhance site-specific musculoskeletal tissue regeneration.

Role of direct estrogen receptor signaling in wear particle-induced osteolysis.

Estrogen withdrawal following surgical ovariectomy was recently shown to mitigate particle-induced osteolysis in the murine calvarial model. Currently, we hypothesize that estrogen receptors (ERs) were involved in this paradoxical phenomenon. To test this hypothesis, we first evaluated polyethylene (PE) particle-induced osteolysis in the murine calvarial model, using wild type (WT) C57BL6J female mice, ERα deficient (ERαKO) mice, and WT mice either treated with 17ß-estradiol (E2) or with the ER pan-antagonist ICI 182,780. According to micro-CT and histomorphometry, we showed that bone resorption was consistently altered in both ERαKO and ICI 182,780 treated mice as compared to WT and E2 groups. Then, we demonstrated that ER disruption consistently decreased both PE and polymethylmethacrylate (PMMA) particle-induced production of TNF-α by murine macrophages in vitro. Similar results were obtained following ER blockade using ICI 182,780 in RAW 264.7 and WT macrophages. ER disruption and pre treatment with ICI 182,780 resulted in a consistent down-regulation of particle-induced TNF-α mRNA expression relative to WT macrophages or untreated RAW cells. These results indicate that the response to wear particles involves estrogen receptors in female mice, as part of macrophage activation. Estrogen receptors may be considered as a future therapeutic target for particle-induced osteolysis.