JPC SYSTEMIC PATHOLOGY
Signalment (JPC #1505161): 4-year-old female dog
HISTORY: The dog was slightly dehydrated, had a left head tilt, and forelimb paresis.
HISTOPATHOLOGIC DESCRIPTION: Slide A: Brain stem: Effacing approximately 30% of the brainstem, affecting both gray and white matter, are multifocal to coalescing granulomas up to 1.5mm in diameter composed of a central area of lytic necrosis surrounded by high numbers of epithelioid macrophages, lymphocytes, plasma cells, few neutrophils, and occasional multinucleated foreign-body type giant cells. Within these granulomas are numerous extracellular and intrahistiocytic, 5-15 um diameter, round, pale, yeasts that have a 1 um thin wall surrounded by a 2-8 um thick, amphophilic, mucinous capsule and occasionally exhibit narrow-based budding. The neuroparenchyma adjacent to the granulomas is multifocally infiltrated by low numbers of lymphocytes, contains increased numbers of glial cells (gliosis), and multifocally displays increased pallor (rarefaction) and vacuolation (spongiosis). Multifocally, there is expansion of the Virchow-Robin space by moderate numbers of lymphocytes, plasma cells, and occasional macrophages (perivascular cuffing) and hemorrhage, fibrin, and/or edema. Multifocally, there is expansion of the leptomeninges by moderate numbers of lymphocytes, plasma cells, and macrophages.
Slide B: Mucicarmine stain: Yeast capsules are diffusely carminophilic, which multifocally highlights yeast that exhibit narrow-based budding.
MORPHOLOGIC DIAGNOSIS: Brain stem: Granulomas, multifocal to coalescing, marked, with mild lymphohistiocytic meningitis, perivascular cuffing, and numerous extracellular and intrahistiocytic carminophilic yeast, etiology consistent with Cryptococcus neoformans, breed not specified, canine.
ETIOLOGIC DIAGNOSIS: Cryptococcal meningoencephalitis
CAUSE: Cryptococcus neoformans var. neoformans
SYNONYMS: European blastomycosis (torulosis)
· Dimorphic, saprophytic, yeast-like fungus with worldwide distribution, predilection for respiratory and central nervous systems
· Infection is neither contagious nor zoonotic
· Most cases caused by one of two species:
· C. neoformans: Worldwide distribution; usually infects immunocompromised (T cell deficient) hosts; pigeons regarded as the most important vector; the pigeon’s high body temperature may protect it from infection; found in pigeon or other avian guano and soil; affects primarily immunocompetent cats, and less likely dogs, horses, cattle, goats, NHPs, ferrets, guinea pigs, various exotic species, and immunocompromised humans; two varieties:
· C. neoformans var. grubii: The most common isolate worldwide from humans and animals with cryptococcosis
· C. neoformans var. neoformans
· C. gattii (formerly C. neoformans var. gattii): Historically restricted to tropical/subtropical climates; cause of an ongoing outbreak in the North American Pacific Northwest involving humans, cats, dogs, and other animals; occurs most often in immunocompetent hosts; several species of eucalyptus trees provide an environmental niche; koalas are sentinel hosts; more frequent in cats than dogs; more likely to be a primary pathogen without underlying immune deficiencies
· The only pathogenic fungus that secretes a polysaccharide capsule
· Organism is unencapsulated in the environment (present in soil and bird [especially pigeon] guano) > aerosolization of basidiospores or desiccated yeast cells > infection (usually via inhalation):
· Nasal cavity is primary site of infection in cats, dogs, koalas, and psittacines; infection may spread locally from the nasal cavity to involve the skin, oral mucosa, eyes or brain
· Lungs are primary site of infection in humans but pulmonary involvement usually mild/asymptomatic
· In the host body, organism regains capsule > may disseminate hematogenously or via lymphatics or extend locally from the nasal cavity to the CNS
· Multifocal cutaneous involvement results from hematogenous dissemination in most cases; rarely may develop localized cutaneous disease due to penetrating skin injury
· Virulence factors:
· Mucopolysaccharide (glucuronoxylomannan) capsule:
· Prevents phagocytosis by macrophages and inhibits leukocyte migration and recruitment of inflammatory cells
· Phenotypic switching in size and structure of capsular polysaccharides may enhance immune response evasion
· Laccase (major virulence factor) catalyzes the formation of a melanin-like pigment which has antioxidant properties
· Serine proteinase cleaves fibronectin and other basement membrane proteins; may aid in tissue invasion
· Urease expression may promote sequestration in microcapillaries, a critical step in dissemination to the brain
· Immunity is cell-mediated and dependent on delayed-type hypersensitivity reactions: IFN-y and other cytokines elicit and activate macrophage +/- neutrophils to kill the yeast by using reactive nitrogen and oxygen intermediates
· Immune suppression of cell-mediated immunity (FIV or FeLV in cats, Ehrlichia canis or long-term glucocorticoid therapy in dogs) appears to increase susceptibility
TYPICAL CLINICAL FINDINGS:
· Majority of clinical signs are attributable to infection of the primary organ:
· CNS: Depression, seizures, circling, head pressing, paresis, blindness (encephalitis)
· Skin: Multifocal papules and nodules ranging from 1-25 mm in diameter, +/- ulceration
· Upper respiratory tract: Sneezing, snuffling, mucopurulent to hemorrhagic chronic nasal discharge; +/- swelling over bridge of nose that may progress to facial deformity; +/- otitis interna, dyspnea, mandibular lymphadenomegaly
· Eyes: Peripheral blindness with unresponsive pupils (chorioretinitis or panopthalmitis)
TYPICAL GROSS FINDINGS:
· Multiple small “cysts” (“cryptococcomas”) with a viscous, gelatinous appearance (not true cysts – are filled with microbes and the faint gray appearance is caused by the mucinous capsules)
· Large gelatinous lesions (torulomas,) caused by unrestricted growth
· Increased meningeal opacity with edema and congestion
· Skin: Lesions (mainly of nose and head) are usually small, firm nodules that ulcerate, exude a small amount of serous exudate, then heal
· Upper respiratory tract: Granulomatous rhinitis +/- facial deformity, swelling over nasal plane
· Eyes: Optic neuritis, chorioretinitis
· Other: Renal granulomas, lymphadenomegaly with gelatinous appearance, otitis interna (C. neoformans var. grubii), mastitis, placentitis with abortion and infection of the fetus
TYPICAL LIGHT MICROSCOPIC FINDINGS:
· Pleomorphic, round or oval, thin‑walled cells (2-10 um diameter) surrounded by a thick mucopolysaccharide capsule (1-30 um thick) that forms a clear or refractile halo
· Organisms with unstained capsules create “soap-bubble” appearance
· Reproduce by narrow-based budding
· Leptomeninges: loosely organized, lacy appearance with often myriad organisms
· Inflammation usually scant (especially in cats), but may be mild to moderate and consist of macrophages with engulfed organisms and variable numbers of neutrophils, eosinophils, and mononuclear cells; leptomeningeal inflammation can extend along the roots of cranial nerves; inflammatory response is often more intense in the dog
· Rare granulomas with occasional multinucleate giant cell macrophages
· Ocular involvement: Organisms between the choroid and retina
· Characteristic filamentous capsule: thin filaments are anchored in the fungal cell wall and radiate peripherally, where they are thickened at the periphery by interstitial protein
· Other features: distinct nucleus, prominent nucleolus, ribosomes, dense granule, mitochondria, laminated cell wall with an electron lucent zone and reticulated capsule
· Microbody (cytoplasmic organelle): a modified peroxisome involved in xanthine and urate metabolism; may underlie propensity to live in urate-rich bird guano
ADDITIONAL DIAGNOSTIC TESTS:
· Histochemical stains:
· Mucopolysaccharide capsule stains positively by Mayer’s mucicarmine or Alcian blue; negative staining of wide capsular zone with India ink
· Yeasts stain with PAS or GMS
· Latex cryptococcal agglutination test (LCAT): Measures cryptococcal polysaccharide capsular antigen in serum or cerebrospinal fluid; rapid and very sensitive
· Fungal culture (may take up to 6 weeks)
· PCR: Detection of CAP59 gene
· IHC: Positive results are only suggestive, and should be supported by other tests
· CT/MRI: Findings may be suggestive, e.g. case report in a koala with atypical presentation (Martinez-Nevado, Jour Wildlife Zoo Med 2017)
For microscopic findings: Poorly encapsulated C. neoformans is difficult to differentiate from other unencapsulated fungi
· Blastomyces dermatitidis (I-F06, P-F05, S-F02): lacks a thick capsule; exhibits broad-based budding
· Histoplasma capsulatum var. capsulatum (D-F01, P-F02, N-F01): smaller (2-5 um); usually within macrophages
· Coccidioides immitis (C-F01, P-F03): much larger (5-50 um); reproduces by endosporulation rather than budding
· Candida parapsilosis (cat): granulomatous rhinitis; yeast and hyphal forms in tissue
· Horses: Cryptococcosis may cause rhinitis, pneumonia, meningitis, encephalitis, abortion with mycotic placentitis, fetal pneumonia, and disseminated infection
· Donkey: Skin lesions
· Goats: Mastitis, pulmonary infection, meningoencephalitis
· Cattle: Mastitis, pulmonary infections, meningoencephalitis (var. grubii)
· Sheep: Mastitis and rhinitis
· Water buffalo: Mastitis
· Guinea pigs (laboratory): Multifocal granulomatous lesions in the meninges with fibrosis; focal cutaneous lesions
· Cheetahs, free-ranging and captive: Ulcerative skin nodules and gelatinous foci in several tissues (C. gattii)
· Apes and gorillas: Affected with both C. neoformans and C. gattii; cloudy meninges and cystic degeneration of the periventricular white matter
· Koalas: Sentinel animals for C. gattii; primarily targets lower respiratory tract but can be disseminated to the CNS
· Kangaroo: Case report of granulomatous pneumonia and severe bilateral glomerular and tubulointerstitial amyloidosis inducing protein-losing nephropathy (Thurber, et al. Jour Wildlife Zoo Med 2017)
· Dolphins/porpoises: Progressive disease starting as mild lymphoplasmacytic inflammation in the lungs, followed by nodular to diffuse granulomatous inflammation, and ending with multisystemic fungemia
1. Barthold SW, Griffey SM, Percy DH. Pathology of Laboratory Rodents and Rabbits. 4th ed. Ames, IA: Wiley-Blackwell; 2016:79, 234-235.
2. Caswell JL, Williams KJ. Respiratory system. In: Maxie MG, ed. Jubb, Kennedy, and Palmer’s Pathology of Domestic Animals. Vol 2. 6th ed. St. Louis, MO: Elsevier, Inc: 2016:582-583.
3. Cheville NF. Ultrastructural Pathology. 2nd ed. Ames, IA: Wiley-Blackwell; 2009:585-586.
4. Ellison D, Love S. Fungal infections. In: Neuropathology. Barcelona, Spain: Mosby International Ltd; 1998:17.7-17.9.
5. Headley SA, et al. Pathologic, histochemical, and immunohistochemical findings in pulmonary and encephalitic cryptococcosis in a goat. Jour Vet Diagn Invest. 2019 Jan;31(1):69-73.
6. Higgins D, Rose K, Spratt D. Monotremes and Marsupials. In: Terio KA, McAloose D, St. Leger J, eds. Pathology of Wildlife and Zoo Animals. San Diego, CA: Elsevier; 2018: 471.
7. Kaufman-Francis K, et al. The Early Innate Immune Response to, and Phagocyte-Dependent Entry of, Cryptococcus neoformans Map to the Perivascular Space of Cortical Post-Capillary Venules in Neurocryptococcosis. Am Jour Pathol. 2018 Jul;188(7):1653-1665.
8. Leger JS, Raverty S, Mena A. Cetacea. In: Terio KA, McAloose D, St. Leger J, eds. Pathology of Wildlife and Zoo Animals. San Diego, CA: Elsevier; 2018: 561-562.
9. Lowenstine LJ, McManamon R, Terio KA. Apes. In: Terio KA, McAloose D, St. Leger J, eds. Pathology of Wildlife and Zoo Animals. San Diego, CA: Elsevier; 2018: 397.
10. Martinez-Nevado E, et al. Atypical presentation of Cryptococcus neoformans in a koala (Phascolarctos cinereus): A magnetic resonance imaging and computed tomography study. Jour Zoo Wildl Med. 2017:48(1): 250-254.
11. McAdam AJ, Milner DA, Sharpe AH. Infectious diseases. In: Kumar V, Abbas AK, Aster JC, eds. Robbins and Cotran Pathologic Basis of Disease. 9th ed. Philadelphia, PA: Elsevier Saunders; 2015:387-388.
12. Miller AD and Zachary JF: Nervous system. In: Zachary JF, ed. Pathologic Basis of Veterinary Disease. 6th ed. St. Louis, MO: Mosby, Inc.; 2016:841-843.
13. Olszewski MA, Noverr MC, Chen G-H, Toews GB, Cox GM, Perfect JR, Huffnagle GB. Urease expression by Cryptococcus neoformans promotes microvascular sequestration, thereby enhancing central nervous system invasion. Am Jour Pathol. 2004;164(5):1761-1771.
14. Paulin J, Morshed M, and Armien JG. Otitis interna induced by Cryptococcus neoformans var. grubii in a cat. Vet Pathol. 2012;50(2):260-263.
15. Riet-Correa F, Krockenberger M, Dantas AFM, Oliveira DM. Bovine cryptococcal meningoencephalitis. Jour Vet Diag Inv. 2011;23(5):1056 –1060.
16. Schmertmann LJ, et al. Identification of the environmental source of infection for a domestic ferret with cryptococcosis Jour Vet Diagn Invest. 2019 Nov;31(6):828-835.
17. Summers BA: Inflammatory diseases of the central nervous system. In: Cummings JF, de Lahunta A, eds. Veterinary Neuropathology. St. Louis, MO: Mosby-Year Book, Inc.; 1995:151-155.
18. Sykes JE, Malik R. Cryptococcus. In: Greene CE, ed. Infectious Diseases of the Dog and Cat. 4th ed. St. Louis, MO: Elsevier Inc; 2012:621-634.
19. Terio KA, McAloose D, Mitchell E. Felidae. In: Terio KA, McAloose D, St. Leger J, eds. Pathology of Wildlife and Zoo Animals. San Diego, CA: Elsevier; 2018: 278.
20. Thurber MI, Gjeltema J, Sheley M, Wack RF. Cryptococcus neoformans var. grubii-associated renal amyloidosis causing protein-losing nephropathy in a red kangaroo (Macropus rufus). J. of Zoo and Wildlife Med. 2017:48(3): 929-932.