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: Brain stem: Disrupting the gray and white matter are multifocal to coalescing granulomas composed of a central area of necrosis surrounded by high numbers of epithelioid macrophages, lymphocytes, plasma cells, low numbers of neutrophils, and occasional multinucleated foreign-body type giant cells. Within these foci are numerous extracellular and intrahistiocytic, 5-15 um diameter, round, pale, extracellular yeasts that are characterized by a 1 um thin wall; surrounded by a 2-8 um thick, amphophilic, mucinous capsule; and occasionally exhibit narrow-based budding. Low numbers of lymphocytes multifocally infiltrate the adjacent neuropil. Multifocally, there is expansion of the Virchow-Robin space by moderate numbers of lymphocytes, plasma cells and occasional macrophages (perivascular cuffing) and hemorrhage, fibrin, 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, and the stain multifocally highlights yeast that exhibit narrow-based budding.
MORPHOLOGIC DIAGNOSIS: Brain stem: Meningoencephalitis, granulomatous, multifocal to coalescing, marked, with 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
· Dimorphic, saprophytic, yeast-like fungus with worldwide distribution, found in pigeon droppings and soil; affects primarily immunocompetent cats, dogs, horses, and immunocompromised humans; predilection for respiratory and central nervous systems
· 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; two varieties:
o C. neoformans var. grubii: The most common isolate worldwide from humans and animals with cryptococcosis
o 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, 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
· 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 body, organism regains capsule > maydisseminate 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:
· Polysaccharide (glucuronoxylomannin) 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
· Capsular genes CAP 59 and CAP64 associated with virulence
· 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
· Predilection for CNS may be because the cerebrospinal fluid lacks alternative pathway complement components found in serum that would bind to the carbohydrate capsule and enhance phagocytosis and killing by neutrophils
· Adaptive immune response: Toll like receptor 9 (TLR9) signaling
· 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
· Skin: Multifocal papules and nodules ranging from 1-25 mm in diameter, +/- ulceration
· Upper respiratory tract: Sneezing, snuffling, mucopurulent to hemorrhagic nasal discharge; +/- swelling over bridge of nose that may progress to facial deformity; +/- otitis media, dyspnea, mandibular lymphadenomegaly
· Eyes: Peripheral blindness with unresponsive pupils
TYPICAL GROSS FINDINGS:
· Multiple small “cysts” 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 media (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 heteropolysaccharide 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:
· Mucopolysaccharide capsule stains positively by Mayer’s mucicarmine or Alcian blue; organism stains with PAS or GMS
· India ink: Negative staining of wide capsular zone
· 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); cytology
· PCR: Detection of CAP59 gene
· IHC: Positive results are only suggestive, and should be supported by other tests
For microscopic findings:
· Poorly encapsulated C. neoformans is difficult to differentiate from other unencapsulated fungi
· Blastomyces dermatitidis: lacks a thick capsule; exhibits broad-based budding
· Histoplasma capsulatum var. capsulatum: smaller (2-5 um); usually within macrophages
· Coccidioides immitis,: much larger (5-50 um); reproduces by endosporulation rather than budding
· Candida parapsilosis (cat): granulomatous rhinitis; yeast and hyphae
· Horses: Cryptococcosis may cause rhinitis, pneumonia, meningitis, encephalitis, abortion with mycotic placentitis, fetal pneumonia, and disseminated infection
· Donkey: Skin
· Goats: Mastitis, pulmonary infection, meningoencephalitis
· Cattle: Mastitis, pulmonary infections, meningoencephalitis (var. grubii)
· Sheep: Mastitis and rhinitis
· Water buffalo: Mastitis
· Neonatal southern right whale: Both hyphae and yeast were cultured from the urine of a neonatal southern right whale (Eubalaena australis) and identified by PCR
· TLR4 mutant mice: Primary receptor for recognition of the capsule of Cryptococcus neoformans
· C3H/HeJ has a point mutation in the Tlr4 gene that renders it unresponsive to LPS
· A less common strain, C57BL10/ScCr, has a different null mutation of Tlr4
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