History

A 6-year-old 40.5-kg castrated male Golden Retriever mixed-breed dog was referred for evaluation of seizure activity, blindness, and inappetence following a 2-month history of intermittent constipation and lethargy. One month prior to referral, the dog had developed skin lesions. At the time of referral, the dog was being treated with oral carprofen, gabapentin, and fluconazole (doses unknown). The dog had no history of clinically relevant illness or injury. It had been transported from Georgia as a puppy and had not traveled outside of New England since.

Clinical and Clinicopathologic Findings

On presentation, the dog was dull and unwilling to stand but able to walk with support. Proprioceptive deficits were noted in both pelvic limbs. Ulcerated cutaneous nodules of varying sizes were appreciated over the entirety of the body (Figure 1). The oral mucosa was multifocally ulcerated, and the mandibular and retropharyngeal lymph nodes were markedly enlarged.

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Images of cutaneous and ocular lesions of a 6-year-old 40.5-kg castrated male Golden Retriever mixed-breed dog evaluated because of seizure activity and signs of blindness and inappetence following a 2-month history of intermittent constipation and lethargy. A—Numerous raised, pink, ulcerated cutaneous nodules of various sizes are evident. Similar lesions multifocally affected the entirety of the body. B—The left eye has multiple conjunctival nodules (asterisks), a superficial corneal ulcer ventrally (arrow), and multiple iridal nodules (arrowheads). C—Photograph of the left fundus taken after pharmacological pupillary dilation, featuring a swollen optic nerve head and multiple subretinal nodules of various sizes (double daggers).

Citation: Journal of the American Veterinary Medical Association 261, 1; 10.2460/javma.22.06.0269

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Menace response and dazzle reflex were absent, and pupillary light reflexes were severely diminished bilaterally. Multiple conjunctival nodules were present, several of which appeared to have abraded the cornea, producing superficial ulcers bilaterally. Trace flare was present bilaterally along with multiple pale nodules in the right iris. Fundic evaluation revealed multiple raised, pale-pink subretinal nodules and swollen hyperemic optic nerve heads bilaterally (Figure 1).

A CBC (ADVIA 2120; Siemens Inc) showed moderate leukocytosis (30.84 X 10³ WBCs/µL; reference interval [RI], 4.4 X 10³ to 15.1 X 10³ WBCs/µL) characterized by mature neutrophilia (22.8 X 10³ cells/µL; RI, 2.8 X 10³ to 11.5 X 10³ cells/µL) and eosinophilia (3.39 X 10³ cells/µL; RI, 0 X 10³ to 1.4 X 10³ cells/µL). A serum biochemical panel (Cobas-6000; Roche Diagnostics) revealed moderate hypoglycemia (52 mg/dL; RI, 67 to 135 mg/dL), mildly low BUN (5 mg/dL; RI, 8 to 30 mg/dL) and creatinine (0.5 mg/dL; RI, 0.6 to 2.0 mg/dL), and hypomagnesemia (1.4 mg/dL; RI, 1.8 to 3.0 mg/dL), hypernatremia (159 mEq/L; RI, 140 to 150 mEq/L), hyperchloremia (118 mEq/L; RI, 106 to 116 mEq/L), and hypoalbuminemia (2.4 g/dL; RI, 2.8 to 4.0 g/dL). Serology (Snap 4Dx; Idexx Laboratories) results were negative for Anaplasma phagocytophilum, Ehrlichia canis, and Borrelia burgdorferi antibodies and Dirofilaria immitis antigen.

Additional Clinical and Clinicopathologic Findings

Fine-needle aspirates from cutaneous lesions on the face and forelimb were submitted for microscopic evaluation and appeared cytologically similar (Figure 2). The preparations were moderately cellular, well preserved, and mildly blood-contaminated on a pale to densely basophilic background with some lysed nuclei and streaming nuclear material. Abundant yeast organisms were present, scattered throughout the smears and frequently found in variably sized clusters. These organisms were round or oval and measured approximately 4 to 8 μm in diameter, with distinct borders and a smooth to wrinkled or folded, variably stained internal structure. These forms were often surrounded by a clear, nonstaining capsule and narrow-based budding was occasionally observed, consistent with Cryptococcus spp. Macrophages were frequently found, often containing phagocytized fungal organisms, along with several inflammatory multinucleated giant cells. Small, well-differentiated lymphocytes, plasma cells, and mature, nondegenerate neutrophils were seen less frequently.

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Photomicrograph of a fine-needle aspirate sample from a facial lesion of the dog in Figure 1. There are numerous oval yeast organisms (arrows) containing a variably thick, nonstaining capsule and occasionally exhibiting narrow-based budding, consistent with Cryptococcus spp. Variably vacuolated macrophages (arrowhead) were frequently seen throughout the preparations. Modified Romanowski stain; bar = 20 µm.

Citation: Journal of the American Veterinary Medical Association 261, 1; 10.2460/javma.22.06.0269

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A serum latex agglutination test (Idexx Laboratories) was performed for Cryptococcus spp antigen and was positive at a 1:32,768 dilution. The dog was subsequently euthanized due to declining condition and poor prognosis. A necropsy was performed.

Gross and Histopathologic Findings

In addition to the dermatologic and ocular findings noted during the antemortem physical examination, necropsy revealed marked lymphadenopathy and multifocal pale nodules in the peripheral lymph nodes, cutaneous trunci and temporalis muscles, lungs, and kidneys (Figure 3). A small volume of red-tinged fluid was present in the pleural space, and mild mucosal hemorrhage was noted in the duodenum to midjejunum.

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Postmortem images of the same dog. A—Marked subcutaneous edema is evident, especially in the periorbital and perimandibular regions. B—Multiple coalescing tan subcutaneous granulomas (arrows) are throughout the subcutis, reflected from the dorsal aspect of the skull, and there is bilateral diffuse atrophy of the temporalis muscles. C—Raised, tan granulomas are seen across all lung lobes, and the tracheobronchial lymph nodes (asterisk) are extremely enlarged. D—The right prescapular lymph node contains coalescing, red to tan nodules that bulged on cut surfaces along the node’s periphery. E—Fungal granulomas are within the cerebellar folia (asterisk) and diffusely throughout the meninges (double daggers). H&E stain; bar = 500 µm. F—A large fungal granuloma (asterisk) effaces and compresses histologically normal thyroid tissue, and multiple smaller fungal granulomas are present. H&E stain; bar = 500 µm. G—A subretinal fungal granuloma (asterisk) and infiltration surround the optic nerve (another manifestation of meningitis) resulting in retinal detachment and clinical blindness. H&E stain; bar = 500 µm. H—Yeast organisms within a prescapular lymph node have intense aquamarine staining of their thick, mucopolysaccharide capsules. Alcian blue stain; pH = 2.5; bar = 50 µm.

Citation: Journal of the American Veterinary Medical Association 261, 1; 10.2460/javma.22.06.0269

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Microscopically, disseminated granulomas with intralesional yeast were present in the skin, subcutis, cutaneous trunci and temporalis muscles, tongue, spleen, liver, kidneys, lungs, thyroid gland, lymph nodes, conjunctiva, irides, choroid, optic nerves, and brain and meninges (Figure 3). Granulomas were composed of epithelioid macrophages with interspersed multinucleated giant cells and neutrophils and with yeast-like organisms seen intra- and extracellularly. These measured 5 to 7 µm and were round with a thin periodic acid–Schiff stain–positive wall and a thick carminophilic and Alcian blue–positive capsule. Narrow-based budding was noted along with occasional pseudohyphal structures. Morphological characteristics were consistent with Cryptococcus spp.

PCR and Sequencing

A PCR protocol targeting the 18S small subunit rRNA gene,¹ performed on material scraped off the cytologic preparations from skin lesions, yielded an amplification fragment of approximately 380 bp after primer editing. Direct, bidirectional sequencing confirmed the presence of a Cryptococcus sp in the sample, with 100% identity to both Cryptococcus neoformans and Cryptococcusgattii (unable to be differentiated on the basis of the amplified region).

Morphologic Diagnosis and Case Summary

Morphologic diagnosis: disseminated granulomatous inflammation (eyes, brain, lungs, liver, spleen, kidneys, muscle, thyroid gland, lymph nodes, and skin) with intralesional encapsulated Cryptococcus spp yeast.

Case summary: disseminated cryptococcosis in an apparently immunocompetent dog.

Comments

The gross and histopathologic findings in this report are consistent with disseminated cryptococcosis. The 2 primary disease-causing species of the encapsulated haploid saprophytic yeast Cryptococcusare C neoformans, found worldwide, and C gattii, found largely in the tropics and subtropics but also reported in parts of Europe and the west coast of North America.^2,3 C gattii has also been identified in samples from a human patient in New England without a contributory travel history.⁴

Cryptococcus typically infects the host via the respiratory tract, later disseminating hematogenously but, in rare cases, can also infect a host via a damaged skin barrier.³ Cryptococcosis is the most common systemic fungal disease in cats but is now considered an emerging systemic mycosis in dogs.⁵ It has also been documented in numerous other mammalian species, including humans.^3,6

Dogs are more likely than cats to have CNS involvement with profound inflammation and widespread disseminated disease, including but not limited to the skin, lymph nodes, nasal cavity, eyes, and various parenchymal organs.⁷ The ocular lesions described in most canine case reports are characterized by unilateral or bilateral granulomatous or hemorrhagic exudative chorioretinitis, with or without retinal detachment.^8–10 Seizure activity is reported in approximately 43% (9/21) of dogs with CNS cryptococcosis.⁷

Early in the disease process, clinical signs may mimic other conditions, and in geographic regions where this agent is not endemic, or when affecting a species in which this disease does not occur frequently, cryptococcosis can be missed. Prognosis is improved when the condition is caught and treated prior to spread to the CNS.³ Because this dog had no history of travel since puppyhood, and because there is a low incidence of cryptococcosis in New England, diagnosis was delayed in this case until the development of cutaneous lesions.

In addition, the dog of this report had no history of underlying immunodeficiency and had not been treated with immunosuppressive medications prior to onset of clinical signs, further decreasing the initial index of suspicion for systemic mycoses. The more common C neoformans is generally considered an opportunistic pathogen.² C gattii, on the other hand, has previously been thought to have the ability to infect immunocompetent individuals, although recent work suggests that many seemingly immunocompetent humans infected with C gattii may in fact have underlying immune deficiencies.¹¹ However, most dogs with cryptococcosis lack obvious evidence of immunodeficiency. Genetic differences in host immunity seem to play a role in the development of disseminated cryptococcosis in otherwise apparently immunocompetent people, and the same may be true for dogs.¹¹

Speciation was not performed in this case due to poor prognosis and election of euthanasia shortly after diagnosis. The PCR assay performed on material from the fine-needle aspirates was able to confirm a Cryptococcus sp in the sample, but speciation protocols (ie, targeting the internal transcribed spacer gene) were unsuccessful, likely due to the nature of the sample. Speciation could also be performed by use of culture on growth media containing specific D-amino acids that enable differentiation between C neoformans and C gattii.¹² Given the interest in C gattii as an emerging pathogen and the minimal documentation of this organism in New England, speciation would have been ideal from a public health standpoint.

This case serves as a reminder that fungal disease should be included as a differential diagnosis in individuals with potentially consistent clinical signs, regardless of geographic region, travel history, or species. These clinical signs may include weight loss, anorexia, altered mental state or change in behavior, coughing, sneezing, epistaxis, stertorous respiration, cutaneous or subcutaneous lesions, seizures, visual deficits, or ocular lesions. The authors emphasize the importance of early sampling of accessible lesions to obtain a timely diagnosis.