Severe Disseminated Necrotizing and Granulomatous Lymphadenitis and Encephalitis in a Dog Due to Sporotrichum pruinosum (Teleomorph: Phanerochaete chrysosporium )

Abstract

A 9-year-old female mixed breed dog presented for an acute onset of anorexia, vomiting, and cough. Initial examination and diagnostics revealed a large multilobular cranial mediastinal mass with unidentified fungal organisms on cytology. The disease progressed in spite of therapy until the dog was euthanized 8 months later. Gross necropsy findings were a large multilobular intrathoracic mass, mild pleuritis, and generalized lymphadenopathy. Histologic evaluation showed granulomatous inflammation and necrosis with numerous 20- to 70-micron, periodic acid–Schiff- and Gomori methenamine silver-positive spherules effacing lymph node parenchyma, as well as severe inflammation within the midbrain. Endosporulation was a common finding, and large numbers of fungal hyphae were also present in affected areas. Ribosomal RNA gene sequencing found 100% identity to published sequences of Phanerochaete chrysosporium, the teleomorph form of Sporotrichum pruinosum. This is the first published report of disease caused by natural infection with this basidiomycete organism in animals.

A 9-year-old spayed female 24.6-kg mixed breed dog was referred to the Small Animal Internal Medicine Service at the Iowa State University Lloyd Veterinary Medical Center with a 10-day history of vomiting, coughing, intermittent tachypnea, anorexia, and lethargy. The referring veterinarian had examined the dog 1 week prior. Complete blood cell count and serum chemistry findings at that time showed elevated total proteins (7.7 g/dl, reference interval [RI]: 5.2–7.1 g/dl), alkaline phosphatase (187 IU/liter, RI: 20–150 IU/liter), and cholesterol (509 g/dl, RI: 132–300 g/dl). Thoracic radiographs demonstrated large soft tissue opaque masses within the mediastinal space (Fig. 1) resulting in displacement of the trachea and primary bronchi. The masses were most consistent with enlarged sternal and tracheobronchial lymph nodes. Differential diagnoses included neoplasia, such as lymphosarcoma, and systemic fungal infection, such as blastomycosis.

Figs. 1, 2. Fungal lymphadenitis, thoracic cavity, dog. Ventrodorsal thoracic radiographs at initial presentation (Fig. 1) and at 7-month recheck (Fig. 2). Mediastinal masses progressively widen the cranial mediastinum (Fig. 1, black arrows) and markedly displace the primary bronchi, finally extending into the caudoventral portion of the mediastinal space (Fig. 2, black and white arrows).

On presentation to the Lloyd Veterinary Medical Center, the dog was quiet and alert and mildly hyperthermic (temperature = 39.6°C), with a normal heart rate (84 beats/minute), slightly increased respiratory rate (54 breaths/minute), mild generalized muscle wasting, and mildly muffled heart sounds. Thoracic ultrasound confirmed a uniformly hypoechoic multilobular mass at the level of the second intercostal space. Cytologic examination of an ultrasound-guided aspirate of the mass demonstrated mild neutrophilic inflammation with deeply basophilic staining organisms approximately 25 µm in diameter. Periodic acid–Schiff staining of these organisms was positive. A urine Blastomyces antigen test submitted at that time was negative. The dog was discharged from the hospital with oral generic itraconazole (5 mg/kg twice daily; Patriot Pharmaceuticals, Horsham, PA) and a 10-day course of prednisone (0.4 mg/kg once daily). A second urine Blastomyces antigen test was submitted approximately 3 weeks later and was also negative. The dog’s appetite and activity level improved; however, coughing persisted. Following 10 days of treatment, medication was briefly changed to ketoconazole (unknown dose or frequency). Itraconazole therapy was eventually reinstituted due to the onset of anorexia.

The dog re-presented to the Lloyd Veterinary Medical Center 5 months later to investigate the persistent cough. The animal had gained 6.6 kg and physical examination was unremarkable. Complete blood cell count revealed a mild leukopenia (5.73 × 10³/µl, RI: 6–17 × 10³/µl) and mild lymphopenia (0.63 × 10³/µl, RI: 1.0–4.8 × 10³/µl). Serum chemistry findings were increased total proteins (7.6 g/dl, RI: 5.2–7.1 g/dl), alkaline phosphatase (164 IU/liter, RI: 20–150 IU/liter), alanine aminotransferase (100 IU/liter, RI: 24–90 IU/liter), cholesterol (367 mg/dl, RI: 132–300 mg/dl), and triglycerides (131 mg/dl, RI: 24–115 mg/dl). Cytologic examination of ultrasound-guided fine needle aspirates of the cranial mediastinal mass revealed blood, lipid, and a low number of epithelial cells. Bacterial and fungal cultures were negative after 10 days’ incubation. IgG and IgM antibody titers to Coccidioides were negative, and ultrasound-guided biopsy of the mass was declined at that time. Recheck radiographs obtained 7 months after initial presentation demonstrated progressive enlargement of the mediastinal masses to involve all portions of the mediastinal space (Fig. 2). One month later, the dog was euthanized due to the lack of response to treatment and submitted to the Department of Veterinary Pathology Necropsy Service at Iowa State University.

On external examination, body condition was adequate with no obvious external lesions observed. The lung lobes and heart were caudodorsally displaced by an approximately 12 × 15 × 8 cm, multilobular, irregular, firm, yellow-tan mass located within the cranioventral chest cavity (Fig. 3) as well as 2 smaller masses (each 7 × 4 cm) expanding and effacing the normal location of the tracheobronchial lymph nodes. The cut surface was diffusely yellow-tan and friable. The pleural surface of the diaphragm and intercostal spaces was variably covered by raised, multifocal to coalescing, tan plaques.

Fig. 3. Fungal lymphadenitis, thoracic cavity, dog. A large, multilobular, yellow-tan cranioventral thoracic mass caudodorsally displaces the lungs, with tan raised plaques on the pleural surface of the diaphragm. Figs. 4, 5. Fungal lymphadenitis, lymph node, dog. Fig. 4. Postmortem impression smear of mediastinal lymph node shows aggregates of fungal hyphae surrounded by numerous round to oval, variably sized, basophilic organisms in a background of necrosis. Hyphae are occasionally septate with parallel walls and exhibit dichotomous branching. Modified Wright’s. Fig. 5. Replacement of lymph node parenchyma by inflammation surrounding lakes of necrotic debris with abundant fungal spherules. Hematoxylin and eosin.

Similar variably sized masses were present ventromedial to the caudal lung lobes, within the pancreas, at the lesser curvature of the stomach, and surrounding the portal vein at its entrance into the liver. Axillary, submandibular, and internal iliac lymph nodes were enlarged with similar appearance on cut surface. The liver was mildly enlarged. No change in symmetry or color was appreciable in the brain. Numerous spherical and hyphal organisms were observed on lymph node impression smears collected during necropsy (Fig. 4). The remainder of the necropsy was unremarkable. Representative samples of all lesions and major organs were placed in 10% neutral buffered formalin. Samples were paraffin embedded, prepared as 6-µm sections, and stained with hematoxylin and eosin.

Sections from the thoracic mass were most consistent with markedly expanded lymph node parenchyma, which had been effaced by large numbers of macrophages and moderate numbers of plasma cells, multinucleate giant cells, and neutrophils. Large irregular and coalescing lakes of necrosis were commonly present within areas of inflammation. Massive numbers of round fungal spherules were present throughout the areas of inflammation and necrosis, measuring 20 to 70 µm in diameter with a 2- to 5-µm-thick outer cell wall (Fig. 5). Spherules often contained several pedunculated structures budding into the center (endospores), and empty cell wall structures were commonly present. The cell wall was often further surrounded by pale eosinophilic, tightly clustered, radiated linear material that did not stain with either periodic acid–Schiff or Gomori methenamine silver stains, in contrast to the underlying organism (Figs. 6, 7). Large aggregates of fungal hyphae were also present and more numerous within areas of necrosis. Hyphae were occasionally septate with parallel walls and showed acute-angle dichotomous branching. Similar changes were present in sections from all grossly identifiable lymph nodes. The midbrain was diffusely infiltrated by large numbers of macrophages and neutrophils with fewer multinucleate giant cells and plasma cells. Similar previously described spherules and hyphae were observed (Fig. 8). Histopathologic examination of the remaining tissue was unremarkable.

Figs. 6, 7. Fungal lymphadenitis, lymph node, dog. Fig. 6. Positive magenta staining of fungal spherules, often displaying endosporulation. Periodic acid–Schiff. Fig. 7. Dark brown staining of spherules, occasionally displaying endosporulation, scattered among large numbers of hyphae admixed with necrotic debris. Gomori methenamine silver. Fig. 8. Fungal encephalitis, midbrain, dog. Large amounts of inflammatory exudate efface the midbrain; the cerebrum (upper left) is unaffected. Inset: Higher magnification of the midbrain showing macrophages, neutrophils, plasma cells, and multinucleate cells associated with spherules and fungal hyphae. Hematoxylin and eosin.

Paraffin-embedded sections of affected lymph node were submitted to the Washington Animal Disease Diagnostic Laboratory for fungal identification by sequencing. Following DNA extraction, the entire internal transcribed spacer (ITS) region of the ribosomal RNA gene (comprising ITS1, 5.8 S, and ITS2) was amplified via polymerase chain reaction (PCR) using universal fungal primers. Direct sequencing of the PCR amplicon found that the sequence most closely matched that of Phanerochaete chrysosporium (100% sequence identity with GenBank #KM277996). Organism identification was confirmed by a second PCR and sequencing of the D1D2 region of the 28 S ribosomal RNA gene.

Phanerochaete chrysosporium is the teleomorph form, or sexual reproductive stage, of a saprophytic fungal species belonging to the phylum Basidiomycota. Members of this phylum are filamentous fungi that reproduce sexually by forming basidiospores. The anamorph, or asexual reproductive form, is named Sporotrichum pruinosum. This organism is also known as white rot fungus and is commonly associated with rotting wood.

To the author’s knowledge, this is the first described case of natural disease involving either form of this organism in veterinary medicine. Several cases of disease attributed to the dual forms of this agent have been described in humans. Granulomatous pulmonary lesions were found in a middle-age woman regularly exposed to mulch, and Phanerochaete chrysosporium was isolated from biopsy samples. ⁶ A single case of cutaneous infection in a severely immunocompromised patient with advanced human immunodeficiency virus infection has also been described. ⁹ Isolation of Sporotrichum pruinosum from humans with systemic mycotic infection and the pathogenicity of the organism following inoculation into laboratory mice has been described. ^5,11

An interesting feature of this case is the presence of hyphae and yeast forms within the same lesion and in multiple organs. A recent case report of Pythium insidiosum and Blastomyces dermatitidis coinfection in a dog described both yeast and hyphae observed on histopathology. ³ However, yeast consistent with Blastomyces was observed only in the lung, and fungal hyphae consistent with Pythium were observed only in the stomach, intestine, and lymph nodes. Sporotrichum pruinosum is able to reproduce through both budding and the formation of hyphae. ¹¹

Various presentations of disease in dogs due to other basidiomycete organisms have been described. Schizophyllum commune was isolated and confirmed via sequencing from a young dog with large nodules on the neck and in the cranial mediastinum. ⁴ Systemic fungal infections in 2 dogs were attributed to Oxyporus corticola, 1 involving osteomyelitis and systemic dissemination ¹ and 1 presenting with generalized lymphadenopathy. ⁷ Sequences consistent with Inonotus tropicalis were identified in a dog with a mediastinal granuloma ¹⁰ and another with severe fungal myocarditis. ⁸ Trichosporon montevideense has been isolated and sequenced from the brain of a dog with granulomatous meningoencephalitis. ²

Investigation into this dog’s medical history found that the owner acquired the dog at 6 months of age, and its lifetime travel history was limited to Nebraska, Iowa, and Illinois. The medical record indicated a very healthy animal, with only intermittent otitis externa and a lipoma removal 3 years before the onset of disease described in this case. The dog often dug around the owner’s firewood pile, which may have served as the source of the infection. No long-term use of immunosuppressive medication was indicated in the medical record prior to the onset of clinical signs.

Based on the presence of numerous fungal organisms and severity of inflammation within the brain stem, neurologic deficits would have been expected in this dog. However, the only sign suggestive of neurologic disease in this animal on initial presentation was mild depression. Normal mentation was also noted 5 months later. The first sign of neurologic disease occurred 1 week prior to euthanasia, when incoordination was noted by the owner. Fungal dissemination to the brain likely occurred late in the course of the disease.

Fungal organisms were observed in this case 8 months prior to euthanasia; however, the organism could not be definitively identified antemortem. Blastomycosis and coccidioidomycosis were suspected at different points during the investigation of this case; microscopic features were not consistent with either organism. Recently developed molecular techniques have shown the ability to identify uncommon pathogenic fungal species and could be useful in cases where definitive microscopic identification proves difficult.

In summary, severe granulomatous inflammation and necrosis were observed within a large mediastinal mass, multiple lymph nodes, and the midbrain of a dog with a history of unidentified fungal infection. Ribosomal RNA sequencing of paraffin-embedded tissue revealed sequences 100% identical to published sequences of Phanerochaete chrysosporium, the teleomorph form of Sporotrichum pruinosum. This is the first report of disease due to this organism in veterinary medicine. Molecular testing may play an integral role in the successful diagnosis of infection with basidiomycete fungal organisms.