TWO CASES OF CRYPTOCOCCUS GATTII INFECTION IN THE RHESUS MACAQUE (MACACA MULATTA)

Abstract

Cryptococcus gattii was diagnosed in two female indoor-housed rhesus macaques. Gross and histopathologic findings included an isolated pulmonary cryptococcoma in a non-SIV infected macaque and disseminated disease centered on the lungs of an SIV-infected macaque. Fungal yeast were positive with special stains and the diagnoses were confirmed with a lateral flow assay and PCR.

INTRODUCTION

Cryptococcus gattii is a basidiomycete yeast-like fungus, with global distribution, and a predilection for the Pacific Northwest, where it is found in soil and trees [1–5]. It is a primary pathogen in immunocompetent people and has been identified as an opportunistic infection in humans with HIV [4–5]. Infection is acquired through inhalation of the basidiospore or desiccated yeast, with replication in the alveoli, followed by hematogenous dissemination [3].

Cases of cryptococcosis have been reported in several species of nonhuman primates, including macaques. These reports either identified Cryptococcus neoformans as the causative agent or did not speciate the cryptococcal isolate [6–14]. The following describes two cases of C. gattii with differing pathologic presentations in the rhesus macaque.

CASE REPORTS

Case 1:

A female rhesus macaque was born and lived in an outdoor corral at the Oregon National Primate Research Center (ONPRC) for 12 years after which she was moved indoors and assigned to a research protocol. At 14 years old, she was vaginally infected with SIVmac239. All experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC) at the ONPRC, which is fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International [15–16].

Previous clinical history included dental extractions, as well as minor protocol-directed procedures. Five days prior to necropsy, the animal presented with partial anorexia and lethargy. Biochemistry revealed hypoproteinemia (4.7 g/dL), hypoalbuminemia (2.4 g/dL), and azotemia (BUN 175.6 mg/dL, creatinine 5.6 mg/dL). A CBC indicated anemia (HCT 23.8%) and thrombocytopenia (113.0 × 10³/uL). The SIV plasma viral load was 61,000,000 copies/mL at necropsy. Euthanasia was elected due to poor prognosis.

At necropsy, the lungs failed to collapse; the parenchyma was pale pink with variably sized hemorrhages (Fig. 1A). The tracheobronchial lymph nodes were enlarged, pale tan, and bulged on cut section. Additional necropsy findings included diffusely pale renal cortices; thickened gastric, cecal, and colonic mucosae; and serous atrophy of fat.

FIGURE 1.

(A) Case 1: the lungs were diffusely expanded, with multifocal hemorrhages. (B) Case 2: the left cranial lung lobe was focally expanded by a single, well-demarcated pale tan to grey nodule.

Microscopic examination of the lungs revealed diffuse pyogranulomatous interstitial pneumonia, with multifocal hemorrhage and a myriad of extracellular and intrahistiocytic fungal yeast (Fig. 2A). Yeast bodies were ~4–8um, with a ~1–20um thick capsule, and narrow based budding. Throughout the lungs, the yeast were identified in veins, arteries, and rarely within larger airways. The tracheobronchial lymph node contained macrophages that filled the subcapsular sinus and infiltrated the adjacent lymph node parenchyma, where they were frequently associated with yeast bodies. Organisms were also identified within the meninges, heart, pancreas, kidney, stomach, and colon where they were accompanied by little to no inflammatory reaction. The yeast were positive with Gomori’s methenamine silver (GMS) (Fig. 2C) and periodic acid-Schiff (PAS) (Fig. 2D) stains. Additionally, the yeast capsules were positive with mucicarmine (Fig. 2E) and melanin production was confirmed with Fontana-Masson (Fig. 2F). Additional microscopic findings included acute renal tubular degeneration and necrosis, gastritis, and typhlocolitis.

FIGURE 2:

(A) Case 1: Pyogranulomatous infiltrates expand the pulmonary interstitium (H&E). Inset: Subgross photomicrograph demonstrated the diffuse distribution. (B) Case 2: Pyogranulomatous infiltrates expanded the pulmonary interstitium and adjacent alveolar spaces (H&E). Inset: Subgross photomicrograph demonstrated a well-demarcated cryptococcoma surrounded by a thick band of fibrous connective tissue. (C) Yeast bodies were approximately 4–8um, with narrow based budding, and were positive with Gomori’s methenamine silver (GMS) and (D) Periodic acid-Schiff (PAS) stains. (E) The thick capsule of the yeast body was mucicarmine positive. (F) Fontana-Masson confirmed the presence of melanin.

Case 2:

A 9 year-old female rhesus macaque born at the ONPRC in outdoor sheltered housing, was moved indoors at 5 years of age and was eventually assigned to a protocol where she received two unsuccessful adoptive transfers of cells (spleen, lymph node, peripheral blood mononuclear cells) from two rhesus macaques infected with SIVmac239. Throughout 13 sampling time points, the SIV plasma viral load was below the detectable threshold (15 vRNA copies/mL) and was considered negative. Previous clinical history included intermittent emesis and thin body condition. Euthanasia was performed at the designated experimental time point.

At necropsy, the left cranial lung lobe was focally expanded by a single, well demarcated, firm, raised, pale tan to grey nodule ~2.0 × 1.5 × 1.0 cm^3 (Fig. 1B), with a slightly gelatinous texture on cut section. Additional necropsy findings included an ovarian remnant and minimal mucosal thickening throughout the stomach and large intestine.

Microscopic examination demonstrated a focally extensive well-demarcated cryptococcoma characterized by a thick peripheral band of fibrous connective tissue surrounding pyogranulomatous inflammation that expanded the pulmonary interstitium and infiltrated the adjacent alveolar spaces (Fig. 2B). Inflammatory cells were admixed with innumerable extracellular and intrahistiocytic fungal yeast as described in Case 1. Additional microscopic findings included minimal to mild chronic inflammation within the stomach, cecum, colon, kidneys, and trachea.

Ancillary diagnostics in both cases included the cryptococcal antigen lateral flow assay (CrAg LFA; Norman, OK), which were both positive for Cryptococcus sp., as well as PCR and sequencing of formalin fixed paraffin embedded lung at the Washington Animal Disease and Diagnostic Laboratory (Pullman, WA), which identified the organism as Cryptococcus gattii.

DISCUSSION

It is not clear how these animals were exposed to Cryptococcus gattii. Speculatively, exposure occurred while they were housed outdoors, resulting in a subclinical carrier status. With Case 1, infection with SIV and resultant immunosuppression is thought to have allowed for the uncontrolled replication of fungal yeast, with dissemination to multiple organs. The clinicopathologic abnormalities were attributed to typhlocolitis and subsequent diarrhea resulting in dehydration and hypoproteinemia. Diarrhea-induced dehydration leading to renal hypoperfusion resulted in acute renal tubular degeneration, necrosis, and severe azotemia. Inflammation within the intestinal tract and subsequent diarrhea resulted in hypoproteinemia. Case 2 was considered immunocompetent having failed to become SIV-infected after two adoptive transfers. In this animal, the unimpaired immune response limited infection to the lung, where it was ultimately isolated by a thick band of fibrosis forming a cryptococcoma.

While Cryptococcus sp. is not considered directly contagious, there was concern for possible spread via fomites to remaining cohort of SIV-infected animals housed in the same room as Case 1. The animals in this cohort were frequently sedated on the same day for protocol directed blood draws, lymph node biopsies, and bronchoalveolar lavages. The cryptococcal antigen lateral flow assay confirmed that the remaining animals were not subclinically infected.

To our knowledge, this is the first report of C. gattii in the rhesus macaque. These cases highlight the need to consider C. gattii as a potential opportunistic infection in SIV-infected macaques and as an incidental finding in macaques that have previously been housed outdoors.