Case One

A 4 yr old female spayed Labrador retriever was referred to the University of Tennessee College of Veterinary Medicine for lethargy, anorexia, and weight loss of 2 mo duration. The dog appeared normal on physical examination except for a temperature of 103.5°F. Abnormalities on the complete blood count (CBC) included mild anemia (hematocrit [HCT] 39.6%, reference range 41–60%), neutrophilia (17,564 cells/μL, reference range 2,650–9,800 cells/μL), and monocytosis (2,370 cells/μL, reference range 165–850 cells/μL). On a biochemical profile, abnormalities included hyperproteinemia (9.5 g/dL, reference range 4.0–6.8 g/dL) with hypoalbuminemia (2.2 g/dL, reference range 2.9–4.1 g/dL) and hyperglobulinemia (7.3 g/dL, reference range 1.9–3.5 g/dL). Urine-specific gravity was 1.016, and no abnormalities were noted on urine sediment examination.

On the thoracic radiographs, there was a large soft tissue mass in the cranial mediastinum causing marked dorsal and mild rightward deviation of the trachea at both the level of and cranial to the heart base. On a precontrast thoracic computed tomography, the cranial mediastinal mass demonstrated soft tissue attenuation and heterogeneous hyperattenuating areas suggestive of mineralization or acute hemorrhage. The mass caused severe deviation of the trachea and bronchial bifurcation toward the right, wrapping lateral and slightly dorsal to the trachea at the level of the base of the heart.

To further evaluate the cranial mediastinal mass, a thoracotomy was performed. The chest was entered through a median sternotomy, and the mass was deemed not surgically resectable. A 1 cm incision was made in the mass, and a purulent, caseous material was obtained for cytology and culture. Cytologic evaluation showed the sample to be highly cellular with poorly preserved neutrophils and several clear-to-light blue, round, yeast-like forms, which were often surrounded by neutrophils. The yeast measured 8–14 μm in diameter and occasionally appeared to have a refractile cell wall. Examination after periodic acid-Schiff staining revealed many round, bright fuchsia forms of similar size, occasionally containing structures suggestive of budding. The cytological diagnosis was mycosis, with marked suppurative to pyogranulomatous inflammation and necrosis. The dog had an uncomplicated recovery from surgery, and therapy with itraconazole (2.5 mg/kg per os [PO] q 12 hr) was initiated at the time of discharge, 4 days after surgery.

Fungal cultures were performed from the cranial mediastinal mass to identify the infecting mycosis, but the fungus did not match the phenotypic descriptions of any of the hyaline or melanized fungi known at the time. DNA sequencing was therefore performed on the isolate, and the sequences were compared with those stored at the University of Alberta’s Microfungus Collection and GenBank. The final report stated that the phenotypic appearance and DNA sequences of the isolate most closely resembled those of a Sagenomella or Paecilomyces. Two years later, the DNA sequence from the isolate was reevaluated; it matched the sequences from the newly described P caninus.³

Seven months after surgery and antifungal therapy, the dog’s HCT, neutrophil count, and serum globulin concentration were within reference ranges. Fifteen months after the thoracotomy, the dog was evaluated for difficulty rising and neck pain. On thoracic radiographs, there was a persistent, but considerably smaller, soft tissue mass in the cranial mediastinum and a focal interstitial pulmonary pattern in the right cranial lung lobe. Radiographs of the vertebral column showed evidence of early discospondylitis with the presence of indistinct margins and associated underlying sclerosis at the C2–3, C3–4, and C7–T1 vertebral endplates. Carprofen (2.2 mg/kg PO q 12 hr) and tramadol (5 mg/kg PO q 8 hr) were prescribed for pain management. The itraconazole therapy was continued at the same dosage.

Two years after surgery, blood was submitted for a CBC and serum biochemistry profile, and the results were within reference ranges. Thoracic radiographs showed no evidence of a cranial mediastinal mass, but the focal interstitial pattern persisted in the right cranial lung lobe. Cervical radiographs showed persistent narrowing of the previously affected intervertebral disc spaces, with new bone proliferation ventral to all sites that was smooth, opaque, and chronic. End plate lysis at C7–T1 was less pronounced, and there was increased sclerosis, suggesting resolving discospondylitis. Since the HCT, neutrophil count, and serum globulin concentration had normalized, the cranial mediastinal mass had disappeared, and spinal radiographs were consistent with resolving discospondylitis, trial discontinuation of itraconazole therapy was elected. Four and a half years after surgery, the dog was doing well clinically with only occasional episodes of neck pain that were managed with carprofen (2.2 mg/kg PO q 12 hr) and tramadol (5 mg/kg PO q 8 hr).

Case Two

A 3 yr old male castrated cocker spaniel presented to CARE Veterinary Center Specialty Center in Frederick, Maryland, for a 4 mo history of suspected pain, progressive right forelimb lameness, and lethargy. The dog had been adopted as a young adult ∼3 mo prior to the onset of clinical signs. His travel history included Texas and Kansas, and he was living in Washington, DC. Upon physical examination, there were pyrexia (103.2°F), a stiff, short-strided gait, hepatosplenomegaly, and normal-sized but firm prescapular, popliteal, and mandibular lymph nodes. Additionally, the dog would vocalize when touched, which was interpreted as the presence of diffuse pain. The CBC results noted mild anemia (HCT 33 %, reference range 41–58%) and monocytosis (2,059 cells/μL, reference range 165–850 cells/μL). A biochemistry profile showed hypoalbuminemia (1.5 g/dL, reference range 2.9–4.1 g/dL) and hyperglobulinemia (7.8 g/dL, reference range 1.9–3.5 g/dL). The serum was submitted for electrophoresis, and a polyclonal gammopathy was found.

Radiographs of the right humerus showed a lytic lesion of the proximal- to mid-humerus with a sclerotic rim and mild periosteal reaction. Histopathological evaluation of a biopsy of the lesion revealed reactive periosteal bone. A sample of the bone lesion was submitted for aerobic bacterial and fungal culture and was negative. Serum antigen tests for histoplasmosis, blastomycosis, coccidiomycosis, and aspergillosis^a were negative. The tracheobronchial lymph nodes were enlarged on thoracic radiographs. On an abdominal ultrasound, the liver and mesenteric lymph nodes were enlarged, and the spleen was mottled, enlarged, and contained multiple hypoechoic nodules.

Fine-needle aspirations of the spleen were performed using a 22 gauge 1.5 in. needle. Cytological examination of the aspirates showed small-to-moderate numbers of macrophages that occasionally contained yeast structures and pseudohyphae. The fungal organisms had a deep magenta internal structure and a thin, nonstaining cell wall (Figure 1). The yeast forms exhibited narrow-based budding and were approximately 2–5 μm in diameter. Because of the suspicion of an atypical fungal organism, samples were submitted to the University of Tennessee College of Veterinary Medicine’s Microbiology Laboratory. Fungal culture and polymerase chain reaction results were consistent with P caninus. Therapy with itraconazole (5 mg/kg PO q 12 hr) and terbinafine (10 mg/kg PO q 12 hr) was initiated.

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Six weeks after initiation of therapy, the dog’s gait and energy level had improved significantly. Anemia and monocytosis had resolved, and the liver and spleen appeared to be of normal size on abdominal ultrasound. However, hypoalbuminemia (1.8 g/dL) and hyperglobulinemia (5.4 g/dL) persisted. No further diagnostic tests were performed due to financial limitations. At the time of writing, 26 mo after diagnosis, the dog was clinically normal, and the serum albumin and globulin concentration had stabilized. The itraconazole and terbinafine were continued.

Case Three

A 5 yr old male castrated Labrador retriever was presented to the Auburn University College of Veterinary Medicine University College of Veterinary Medicine for evaluation of a 1 mo history of weight loss, hyporexia, and lethargy. Prior travel history included northern Georgia and northern Florida. Upon physical examination, the dog had generalized peripheral lymphadenopathy. Large intra-abdominal masses were palpated caudal to the kidneys, consistent with abdominal lymph nodes. On the CBC, there was an eosinophilia (1704 cells/μL, reference range 100–750 cells/μL). The serum biochemistry analysis identified hyperproteinemia (7.51 g/dL, reference range 5.1–7.3 g/dL) with high-normal albumin of 3.4 g/dL (reference range 2.6–3.5 g/dL) and normal globulins 4.1 g/dL (reference range 2.6–5 g/dL), azotemia (blood urea nitrogen [BUN] 47.4 mg/dL, reference range 10–25 mg/dL; creatinine 3.5 mg/dL, reference range 0–1.3 mg/dL), and hypercalcemia (14.5 mg/dL, reference range 9.5–11.8 mg/dL). The urine-specific gravity was inappropriate at 1.014, which may have been associated with renal azotemia but could also be attributed to hypercalcemia interfering with urine concentrating ability. No abnormalities noted on urine sediment examination. Serum ionized calcium concentration was also elevated (1.51 mg/dL, reference range 1.06–1.28 mg/dL).

On thoracic radiographs, there was sternal and hilar lymphadenopathy with a normal cardiac silhouette, pulmonary vessels, and lung parenchyma. The sublumbar, mesenteric, and hepatic lymph nodes were markedly enlarged and hypoechoic on abdominal ultrasound examination. The spleen was markedly enlarged and had a coarse and lacy echogenicity consistent with infiltrative disease. Small areas of mineralization were present in the cortex of both kidneys. The liver was diffusely hyperechoic.

The patient was sedated with dexmedetomidine (375 mcg/m² IV). Fine-needle aspirates of the liver and spleen were obtained with ultrasound guidance using a 22 gauge 1.5 in. spinal needle. Aspirates of both prescapular and both popliteal lymph nodes were obtained. Cytologic examination of the liver aspirate as well as popliteal and prescapular lymph node aspirates revealed marked pyogranulomatous inflammation with many extracellular yeast-like bodies. Multinucleated giant cells were seen in moderate numbers, had occasionally phagocytosing yeast-like cells that were 2–4 μm in diameter, were stained grey-to-blue, and appeared to be surrounded by a thin halo or capsule. Rarely, narrow-based budding of these yeast cells was noted.

Because of the inability to definitively identify the fungal agent based on cytologic characteristics, lymphadenectomy of the right popliteal lymph node was performed under general anesthesia. A portion of the lymph node was placed in formalin and submitted for histopathology, while another portion was submitted as fresh tissue to the University of University of Tennessee College of Veterinary Medicine’s Microbiology Laboratory for fungal culture and DNA sequencing. Abundant growth of a hyaline mold was observed within 48 hr after plating. Microscopically, the fungus consisted of narrow, septate hyphae with short conidiophores and phialides producing single chains of conidiospores (Figure 2), consistent with P. caninus. The identity was confirmed by genetic sequencing. Based on in vitro testing, the organism was susceptible to amphotericin B, miconazole, itraconazole, and voriconazole and was resistant to fluconazole.

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While awaiting results of the culture and histopathology, the dog was hospitalized and treated with IV fluids and gastroprotectants. A 7 day course of oral prednisone (0.5 mg/kg PO q 12 hr) was prescribed for its calciuretic effects and anti-inflammatory properties, should fungal death incite an inflammatory response. The dog was discharged 4 days later with an improved serum creatinine concentration of 2.2 mg/dL and a serum ionized calcium concentration within the reference range (1.27 mg/dL, reference range 1.06–1.28 mg/dL). Itraconazole (10 mg/kg PO q 24 hr), and terbinafine (30 mg/kg PO q 24 hr) were prescribed.

The dog was rechecked 2 mo later. All clinical signs had resolved, and the dog had gained 4.4 kg. On thoracic radiographs, there was a marked decrease in the size of the sternal lymph node and no visibly enlarged hilar lymph nodes. Rechecks were performed approximately every 2–3 mo. The creatinine normalized (1.3 mg/dL) 4 mo after diagnosis. The BUN remained persistently elevated until 13 mo after diagnosis when it normalized (28.5 mg/dL). Urine-specific gravity was consistently >1.030 at rechecks. Hypercalcemia resolved after 4 mo of treatment (total calcium 10.7 mg/dL). At each recheck, the left popliteal lymph node was aspirated, and samples submitted for cytologic examination. Persistent fungal organisms were seen at each visit; however, subjectively fewer yeast-like bodies were detected compared with the initial presentation.

Thirteen months after diagnosis, while still receiving therapy with antifungal medications, the dog developed vestibular ataxia of all limbs, hypermetria of the right forelimb and right hindlimb, and a left-sided head tilt. The original metabolic derangements of hypercalcemia and azotemia were no longer present (total calcium 11.3 mg/dL, creatinine 1.3 mg/dL, BUN 28.5 mg/dL). Additional advanced imaging was declined by the owner. The dog was hospitalized, and, given the suspected progression of disease on itraconazole and terbinafine therapy, a dose of amphotericin B (0.25 mg/kg diluted in 500 mL 5% dextrose in water and infused IV over 6 hr) was administered. Prednisone (1 mg/kg PO q 24 hr) therapy was initiated for suspected systemic mycosis of the central nervous system (CNS). Approximately 2 hr after completion of the amphotericin B infusion, status epilepticus developed. The seizures were refractory to diazepam administration and phenobarbital loading. The dog was placed and maintained on a propofol at a constant rate of infusion (0.5–0.75 mL/kg/min). After 24 hr, the propofol constant rate of infusion was tapered and discontinued. The neurologic status did not improve over the following week despite initiation of medications to decrease intracranial pressure (0.5 mg/kg mannitol IV over 45 min and 2 mL/kg hypertonic saline IV) and administration of two additional amphotericin B infusions (0.5 mg/kg then 0.75mg/kg IV diluted in 500 mL 5% dextrose in water and administered over 6 hr). Euthanasia was elected.

On necropsy, the left cerebral hemisphere was moderately expanded by an unencapsulated, poorly delineated, firm, white-to-tan mass. The right cerebellar vermis, vertebral bodies of T2–T5, liver, kidneys, and abdominal lymph nodes contained similar masses. On histopathologic examination, these masses were determined to be granulomas with multifocal necrosis; a few yeasts that occasionally formed pseudohyphae; and numerous 2–4 μm wide, Grocott’s methenamine silver stain-positive hyphae with nonparallel walls and acute angle, dichotomous branching. P caninus was grown in culture from the white-to-tan masses.