JPC SYSTEMIC PATHOLOGY

INTEGUMENTARY SYSTEM

January 2026

N-F05

Signalment (JPC #1902063): Young, female New Zealand white rabbit

HISTORY: This rabbit developed diarrhea and CNS signs.

HISTOPATHOLOGIC DESCRIPTION: Slide A: Cerebrum, level of hippocampus, and thalamus: Scattered throughout the gray and white matter are small, occasionally perivascular, variably sized aggregates of epithelioid macrophages, activated microglia (rod cells), fewer lymphocytes, plasma cells, and rare heterophils admixed with small amounts of pyknotic and karyorrhectic cellular debris (necrosis). Rarely, within these foci are 30-40 µm in diameter intracellular vacuoles that contain myriad 1x3 µm, ovoid, refractile microsporidian spores (pseudocysts). Adjacent to affected areas there is mild gliosis. Virchow-Robin space and the leptomeninges are expanded by a previously described inflammatory infiltrate and there is a small amount of hemorrhage within the leptomeninges. Vessels are lined by reactive endothelium.

Slide B: Brown and Brenn's Gram stain: Cerebrum, level of hippocampus, and thalamus: Multifocally there are moderate numbers of intracellular gram-positive, 1 x 3 µm, rod-shaped microsporidia within areas of inflammation and occasionally in areas with no cellular reaction (pseudocyst).

MORPHOLOGIC DIAGNOSIS: Cerebrum and diencephalon: Meningoencephalitis, lymphohistiocytic, chronic, multifocal, moderate, with intracellular, gram-positive microsporidian spores, New Zealand white rabbit (Oryctolagus cuniculus), lagomorph.

ETIOLOGIC DIAGNOSIS: Cerebral encephalitozoonosis

CAUSE: Encephalitozoon cuniculi

ETIOLOGY SYNONYM: Originally known as Nosema cuniculi

GENERAL DISCUSSION:

• An obligate intracellular microsporidian eukaryotic organism that commonly results in latent infection of laboratory rabbits; immunosuppression may lead to clinical disease with neurologic signs and histologic lesions primarily in the brain, eye, and kidney; segmental vasculitis responsible for lesions in many tissues
  • Especially prefers endothelial cells
  • Animals remain permanently infected and excrete the organism in urine
• Currently classified as a fungus within the phylum microsporidia
• Large domestic rabbits often have subclinical infections with incidental renal lesions; Dwarf rabbits are especially susceptible and often have clinical signs including torticollis, uveitis with cataractous change, and disseminated disease

PATHOGENESIS:

• Infection is acquired through ingestion or inhalation, transplacentally, or through iatrogenic transmission via contaminated needles; zoonotic
• Carnivores can be infected by consuming infected rabbit carcasses
• Initial target organs are those of high blood flow, especially lung, liver, and kidney with lesions evident in the brain at three months

LIFE CYCLE:

• Obligate intracellular organism with a simple and direct life cycle
• Spores are shed in urine from infected renal epithelial cells and complete their life cycle in 3‑5 days
• Spores (infective stage) enter host via digestive tract > sporoplasm is released through polar filament and enters susceptible cells (predominantly vascular endothelial cells) via mechanical forces exerted by the extruded polar filament or due to an active migratory process by the sporoplasm > asexual replication occurs to form a sporophorous vacuole > sporoblasts develop into mature spores until the cell ruptures > release of spores into extracellular spaces > spores infect adjacent cells, enter the vascular system, or the renal tubular lumen, may also pass in feces
  • Sporophorous vacuole: Envelope of spore origin
  • Parasitophorous vacuole: Envelop of host origin

TYPICAL CLINICAL FINDINGS:

• Infections usually subclinical; may present with a variety of neurologic signs including head tilt, vestibular signs (circling, nystagmus, rolling), ataxia, and occasionally behavioral changes
• Uveitis and cataracts from lens capsule penetration may be present in young rabbits
• Rarely, placentitis and abortion in a wide range of animals, including horses has been reported

TYPICAL GROSS FINDINGS:

• Usually absent in the CNS
• Eyes- Severe endophthalmitis and cataracts
• Renal lesions (U-P01) – focal, irregular, depressed areas (“pitted appearance”); on cut section, there are indistinct linear, pale, gray-white areas – severe, nonsuppurative interstitial nephritis
• Focal hepatic necrosis and focal myocardial necrosis occasionally present

TYPICAL LIGHT MICROSCOPIC FINDINGS:

• Granulomatous and nonsuppurative inflammation in target organs; tissue changes are most prominent in kidney and brain but organism selectively parasitizes vascular endothelium 🡪 segmental vasculitis
• During early infections, spores may be present in epithelial cells, macrophages, inflammatory foci or free within collecting tubules – in more chronic infections, organisms are harder to find
• CNS: Lesions do not normally occur until 30 days postexposure and most frequently arise in cerebrum; widespread nonsuppurative meningoencephalomyelitis, astrocytosis, and perivascular lymphocytic infiltration; microsporidia within astroglial cells, macrophages, or extracellular within the granulomatous inflammation; meningeal vascular lesions resemble polyarteritis nodosa, become dominated by sclerotic changes
  • Focal gliosis and granulomas surround small vessels; larger vessels with segmental fibrinoid change
• Kidney (U-P01): Focal to segmental nonsuppurative (heavy, almost pure interstitial infiltrates of plasma cells) to granulomatous interstitial nephritis; degeneration of affected epithelial cells at all levels of the renal tubule; fibrinoid necrosis affects some glomeruli; microsporidia within renal tubular epithelium, free within collecting tubules, or in vessel walls
  • arterial lesions resemble those of periarteritis nodosa
• Eye: Phacoclastic uveitis and cataract formation which is believed to follow transplacental infection is common in dwarf rabbits; keratitis, rupture of lens capsule, heterophilic and granulomatous inflammation (MNGS common), periarteritis within the uvea and retina – can see organisms on Gram stains around fragmented lens fibers or w/in macrophages
• Liver: Focal granulomatous inflammation, periportal lymphocytic infiltrates; nonsuppurative portal infiltrations are associated w/ organisms in hepatocytes and Kupffer cells and nodular vasculitis in the triads
• Heart: Multifocal myocardial necrosis and nonsuppurative inflammation, vasculitis; microsporidia in endothelial cells and cardiomyocytes
• Placenta: Necrotizing placentitis, trophoblasts contain large numbers of intracytoplasmic vacuoles filled with microsporidia
• Lung and spleen: focal to diffuse interstitial pneumonitis w/ mononuclear cell infiltration
• Organisms are small (2.5 x 1.5 µm), rod shaped and can be difficult to identify in chronic infections; in acute active infections, microsporidia can be found free in a focus of inflammation; contained within a sporophorous vacuole in endothelial cells, epithelial cells, macrophages, or intracellularly with no associated inflammatory response (pseudocyst)
• A 6-point grading scheme has been described for brain lesions associated with encephalitozoonosis, reflecting the apparent level of organization and chronicity of the lesion (J Vet Diagn Invest, 2024)
  • most frequently observed lesion was the type 4 lesion, comprising a necrotic center, with epithelioid macrophages, gemistocytic astrocytes, occasional multinucleate giant cells, and less prominent lymphocyte infiltration

ULTRASTRUCTURAL FINDINGS:

• Organism is often within a sporophorous vacuole (previously termed “parasitophorous vacuole” until reclassified as a fungus)
• Thick outer coat enclosing a coiled polar filament (polar tube) and one or two nuclei; one end of the filament is attached to the spore wall and the other end is coiled within the spore from 4 to over 30 times depending on the species
• Corrugated, proteinaceous electron dense exospore (spore wall); chitinous radiolucent endospore; anchoring disc at anterior pole; electron lucent posterior vacuole

ADDITIONAL DIAGNOSTIC TESTS:

• Spores are ovid, 1x2-5um acid fast, gram positive and contain a PAS positive polar granule
• Carbol fuchsin (an acid-fast stain) will stain the organisms a distinct purple color
• Can be highlighted with modified trichrome and calcofluor white M2R
• IHC
• Serology, carbon immunoassay test, indirect IFA, dot ELISA, Western blot
• Spores are excreted intermittently in the urine during acute disease

DIFFERENTIAL DIAGNOSIS:

• Histologic differentials: Primary differential is Toxoplasma gondii:

TOXOPLASMA	ENCEPHALITOZOON
Cyst small (60um or less)	Pseudocyst large (up to 120um)
Spores not acid fast	Spores are acid fast
Gram negative, PAS (-)	Gram positive, PAS (+)
Do not stain with carbol fuchsin	Stain with carbol fuchsin
Giemsa: Cytoplasm is granulated	Giemsa: Cytoplasm is light blue
Stains well with H&E	Stains poorly with H&E
Larger, crescent-shaped 2‑6 um	Smaller, rods 1.5 x 2.5 um
Tend to invoke necrosis	Necrosis not a common finding

• Neospora caninum: Difficult to differentiate from Toxoplasma sp.
• Clinical differentials, neurologic signs: Otitis interna, toxoplasmosis, Baylisascaris sp.

COMPARATIVE PATHOLOGY:

• Recently identified Encephalitozoon species of pathogenic importance include E. intestinalis, E. hellem, E. bieneusi, and E. septatai

• E. cuniculi has been reported in a wide range of hosts including mice, rats, muskrats, guinea pigs, hamsters, ground shrews, black-footed ferrets, goats, sheep, pigs, horses, domestic dogs, wild and captive foxes, domestic cats, other carnivores, nonhuman primates, fur seal pups and reptiles
• Dogs, farmed foxes, mink: Subclinical/asymptomatic infection occurs with an unknown prevalence; serious mortalities occur in farmed foxes; Disease is most severe in young animals and can result in death; often due to in utero infection
• Cats: Few reports of E. cuniculi causing lenticular disease in cats
• Birds:
  • E. hellem; reported in a wide range of companion birds, but commonly infects budgerigars and lovebirds; most infections self-limiting and inapparent, but can result in clinical disease with immunosuppression or poor husbandry
    • Pigeons and doves (Columbids) appear to be carriers
    • Psittacine Beak and Feather Disease (I-V04) common associated infection in lovebirds
    • Budgerigar Fledgeling Disease (Polyomavirus) (I-V03, U-V08), cryptosporidia, giardia, Macrorhabdus ornithogaster, and Chlamydia reportedly associated in budgerigars
    • Gross: Lesions primarily in eye, intestines (distended with fluid and/or gas, diarrhea), liver and kidney (irregular pale foci)
      • Infection occurs primarily in enterocytes in the small intestine and response varies from none to chronic inflammation and proliferation
    • Histo: Subacute necrotizing mononuclear hepatitis and lymphoplasmacytic interstitial nephritis; organisms cluster within cytoplasm of infected cells (renal epithelium, hepatocytes, intestinal epithelium, spleen, lamina propria of intestines, ocular conjunctiva), resulting in cytoplasmic expansion and rupture
      • Expansion of intestinal villi due to epithelial cell hypertrophy and identification of intraepithelial organisms is primary histologic finding
  • E. bieneusi genotype D: Fatal infections of falcons in the Middle East
    • Gross: Yellow, well demarcated, raised nodules in liver, kidneys, spleen, intestine
    • Histo: Multifocal necrosis and pyogranulomatous inflammation in the liver, kidney, and spleen; transmural, necrotizing enterocolitis with diphtheritic membrane in the intestine; Organisms are oblong, up to 2 μm long, intracytoplasmic
    • Feces from ducks, cranes, and pigeons may be the source of infection
• NHPs: E. cuniculi and E. bieneusi; limited to New World monkeys, mostly documented in squirrel monkeys, titi monkeys, and tamarins; clinical disease is usually absent or non-specific, but can be multi-systemic, most seriously in the central nervous system; no specific gross lesions
  • E. cuniculi: Granulomatous inflammation and organisms reported in brain, meninges, kidney, lungs, liver, adrenal glands and placenta; vasculitis/perivasculitis is the predominant finding
  • Infected cells in the CNS include endothelial cells, macrophages, and ependymal cells.
  • E. bieneusi: Proliferative non-suppurative cholecystitis and cholangiohepatitis, enteropathy
  • Transplacental transmission documented, resulting in stillbirths and abortions
• Horses: Rare cause of necrotizing placentitis and abortion; organisms within intracytoplasmic vacuoles of trophoblasts
• Captive meerkats: Report of fatal meningoencephalitis (Church, PWZA 2018)
• Guinea Pigs: Subclinical infection as well as multifocal nonsuppurative meningoencephalitis and interstitial nephritis; also subclinicial carriers of E. bienusi
• Mice: Common in pet and fancy mice; rare in contemporary mouse colonies; detected in wild mice (E. bieneusi and E. hellem); multi-systemic disease, ascites, chronic wasting
• Rats: Rare, nonsuppurative focal lesions in the brain, kidneys, and liver

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