An 18-year-old Quarter Horse mare was evaluated at the University of Wisconsin Veterinary Medical Teaching Hospital because of a mass over the right maxillary incisors. The right third incisor had been extracted approximately 18 months earlier after it was fractured by a kick from another horse. The owners reported no problems with the mare's health until they detected a growth in the area of the previous injury just prior to evaluation.
Initial evaluation revealed an ulcerated, firm, darkly pigmented, approximately 5-cm-diameter spherical mass arising from the gingiva lateral and dorsal to the right first to third maxillary incisors (Figure 1). The horse resisted manipulation of the mass. Results of the rest of the physical and oral examinations were unremarkable. Maxillary radiography revealed substantial osteolysis of the roots of the first and second right maxillary incisors (Figure 2). Associated osteolysis in adjacent bone and active aggressive periosteal proliferation of the adjacent dorsal and lateral margins of the right premaxilla were evident. A wedge biopsy of the mass was performed.
Histologic examination of biopsy tissue revealed multiple irregular, well-defined, discrete, black masses (2 to 7 mm in diameter) separated by intense inflammation and fibrosis (Figure 3). Within the black granules was a myriad of fungal hyphae surrounded by black, finely granular, cementlike material. Hyphal elements varied from 4 to 5 μm in diameter and were septate and irregularly branched. Hyphae radiated toward the periphery of the granules and were often bounded peripherally by neutrophils, followed by mixed accumulations of epithelioid macrophages, plasma cells, lymphocytes, and fewer neutrophils enmeshed in a fibrous connective tissue stroma. An occasional finely serrated eosinophilic band along the margins of the black granules was suggestive of Splendore-Hoeppli material. Several lytic bony trabeculae were along 1 margin. The histologic diagnosis was pyogranulomatous stomatitis with multiple eumycotic pigmented fungal granules typical of a black-grain mycetoma.
Surgical excision of the mass was recommended, and treatment was instituted following owner approval. The horse was sedated with xylazine (1.0 mg/kg [0.45 mg/lb], IV) and glycerol guaiacolate (40.0 mg/kg [18.14 mg/lb], IV). General anesthesia was induced with ketamine (2.0 mg/kg [0.91 mg/lb], IV) and maintained with halothane in oxygen in a closed-circle system. Following aseptic preparation and appropriate draping of the mass and surrounding tissue, a full-thickness circumferential incision was made in the mucosa approximately 2.5 cm from the base of the mass. The mass was then excised with a combination of sharp and blunt dissection. Subsequently, the first and second right maxillary incisors were extracted, and the surrounding maxillary bone was curetted thoroughly. Following copious lavage with physiologic saline (0.9% NaCl) solution, the wound was left open to heal by second intention. Tissue was submitted for histologic examination and bacterial and fungal cultures.
Postoperative maxillary radiography confirmed removal of the rostral aspect of the maxilla and right incisors (Figure 4). Persistent heterogeneous opacities within the maxilla that extended caudally and bony proliferation of the dorsal maxillary surface were consistent with fungal osteomyelitis of the remaining rostral portion of the maxilla.
Gross examination of the surgically resected maxillary mass revealed individual black granules ranging from 2 to 7 mm in diameter, although closely associated granules in clusters of 4 to 15 contiguous granules were common (Figure 5). Histologic features were similar to those of the original biopsy specimen. The irregular black granules consisted of fungal hyphae and intervening, finely granular, black material, although some granules had a few intermingled degenerated inflammatory cells. Hyphae were endogenously pigmented, often more conspicuously along the periphery. Large granules had a lamellar appearance and formed discontinuous concentric rings. The orientation of hyphae in a radial pattern along the periphery of the granules was consistently observed.
α-Hemolytic Streptococcus sp and Actinobacillus sp were identified via aerobic bacterial culture of samples from the mass. Fungal culture performed in the Department of Pathology at the University of Texas Medical Branch, Galveston, Tex, resulted in a hyphal mass lacking differential structures after 3 months, which prevented standard phenotypic identification. The fungus was wooly, turned brown after initially being white, and produced a dark brown exudate (Figure 6). The presence of fungal structures in viable tissue confirmed the etiology of the infection. No evidence was found histologically to indicate that the isolated bacteria were pathogenic. All retained tissue samples that were not submitted for culture were embedded in paraffin following formalin fixation, and all fungal culture samples were stored at −80°C. Identification of DNA of the fungus from frozen fungal cultures and contained within paraffin sections was initiated.
The DNA from approximately 1 g of cultured fungal material and from 4 paraffin-embedded sections (10 μm thickness) of the surgically resected mycetoma was extracted and purified by use of a commercially available genomic DNA isolation kita and published methods.1,2 The D1/D2 domains of the LSUP rDNA3 were amplified with proprietary primers D1 and D2.b Polymerase chain reaction was carried out in 50-μL reactions containing 10 μL of template DNA, 2.5 μL (2 pmol) of each primer, and 25 μL of universal mastermix.c The PCR mixtures with either equine DNA or without template were used as negative controls. Polymerase chain reaction assay was performed with a PCR thermal cyclerd under the following conditions: 95°C for 7 minutes, followed by 35 cycles consisting of 95°C for 30 seconds, 50°C for 30 seconds, and 72°C for 30 seconds with a final extension at 72°C for 10 minutes. The PCR products were analyzed via electrophoresis on a 2% agarose gel. Specific 616–base pair fragments were seen for both fungal isolates, whereas no amplicons were apparent in the negative control lanes (Figure 7). The amplified products were purifiedf and subjected to bidirectional sequencing with an automated sequencerg and terminator chemical analysis as recommended by the manufacturer.h The D1 and D2 primers were used to sequence each DNA sample. Sequences were assembled and examined by use of sequencing software.i On the basis of homology searches in the GenBank database, the fungal culture and paraffin section DNA sequences were both determined to be 96% homologous with the LSUP rDNA of Phialophora oxyspora (accession No. AB100630).3
The horse received phenylbutazone (2.0 mg/kg, IV) after recovery and for 2 days after surgery (1.0 mg/kg, PO, q 12 h). Additional postoperative treatment consisted of sodium iodide (1.0 mg/kg, IV) once weekly for 4 weeks and potassium iodide (0.5 mg/kg [0.23 mg/lb], PO, q 24 h) for 14 days. The horse was discharged 10 days after initial evaluation with instructions to monitor the horse for signs of iodide toxicosis, including dry, flaky skin; anorexia; lethargy; or epiphora. Intravenous administration of sodium iodide was performed by the referring veterinarian, and the owners completed oral administration of potassium iodide. One year after evaluation, the owners reported that the mass had not recurred and the surgical site appeared normal.
Large subunit P
BDtract Genomic DNA isolation kit, Maxim Biotech Inc, San Francisco, Calif.
Applied Biosystems, Foster City, Calif.
AmpliTaq Gold PCR Master Mix, Applied Biosystems, Branchburg, NJ.
PTC-200 thermal cycler, Bio-Rad Laboratories, Hercules, Calif.
EZ Load 100–base pair molecular ruler, Bio-Rad Laboratories, Hercules, Calif.
Montage PCR Centrifugal Filter Device, Millipore Corp, Billerica, Mass.
ABI3700 automated sequencer, Applied Biosystems, Branchburg, NJ.
Big Dye Terminator, Applied Biosystems, Branchburg, NJ.
Sequencer, Gene Codes, Ann Arbor, Mich.
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