JPC SYSTEMIC PATHOLOGY

NERVOUS SYSTEM

February 2023

N-P03

 

SIGNALMENT (JPC #2461138): A Saint Bernard

 

HISTORY: The dog presented with a one-week history of ataxia and showed mild obtundation and severe tetraparesis. Postural reactions were decreased to absent in the hindlimbs. Muscle tone was decreased in the hindlimbs and normal in the forelimbs.   The cranial nerve reflexes were within normal limits.

 

HISTOPATHOLOGIC DESCRIPTION: Cerebellum: Multifocally expanding the leptomeninges and Virchow Robin spaces and infiltrating the adjacent neuropil of the molecular layer are numerous macrophages, lymphocytes, fewer plasma cells, and neutrophils admixed with eosinophilic cellular and karyorrhectic debris (necrosis), increased numbers of glial cells (gliosis), and numerous small caliber blood vessels lined by reactive endothelium. Purkinje cells within affected areas are occasionally shrunken, hypereosinophilic with a pyknotic nucleus (necrosis). Multifocally admixed with the inflammatory cells are numerous protozoal cysts and moderate numbers of clusters of free tachyzoites; cysts are up to 70 µm in diameter with a 1 µm thick cyst wall that contain numerous 2x2 µm basophilic bradyzoites, and tachyzoites are 2x6 µm, crescentic, and arranged in small clusters. 

 

Skeletal muscle: Separating, surrounding, and replacing approximately 60% of myocytes are numerous macrophages, lymphocytes, plasma cells, fewer neutrophils, and reactive fibroblasts admixed with hemorrhage, fibrin, and edema. Remaining myocytes have either a pale, swollen, vacuolated sarcoplasm (degenerate), hypereosinophilic, fragmented sarcoplasm with loss of cross-striations and a pyknotic nucleus (necrotic), and rarely basophilic sarcoplasm and multiple nuclei that are internalized and row (regeneration). Scattered within and around muscle fibers are few clusters of previously described protozoal cysts and tachyzoites.

 

MORPHOLOGIC DIAGNOSIS:  

1. Cerebellum: Meningoencephalitis, necrotizing, lymphohistiocytic, multifocal, moderate, with protozoal cysts and extracellular tachyzoites, Saint Bernard, canine. 

2. Skeletal muscle: Myositis, necrotizing and lymphohistiocytic, multifocal, moderate, with myocyte degeneration and regeneration and intra- and extracellular protozoal tachyzoites.

 

ETIOLOGIC DIAGNOSIS: Cerebellar and intramuscular neosporosis

 

CAUSE: Neospora caninum

 

 

GENERAL DISCUSSION: 

• Neospora is a cyst-forming coccidian parasite in the phylum Apicomplexa 
• Neospora is an important cause of abortion and perinatal mortality in cattle and nonsuppurative encephalitis, myocarditis and myositis in dogs
  1. It also causes abortion in sheep, goats, and pigs and is associated with periportal hepatitis in dogs

 

LIFE CYCLE:  

• Similar life cycle to Toxoplasma gondii (N-P02)
• Dogs and wild canids (e.g., wolves and coyotes) are the only known definitive hosts and can also be intermediate hosts; all other animals are intermediate hosts, although herbivores are the most common intermediate hosts
• Three infectious stages: 
  • Oocysts: Present in the definitive host; environmentally resistant, shed in host feces
  • Tachyzoites: Present in intermediate and definitive host; rapidly dividing, found in body fluids (blood) and in cytoplasm of host cells
  • Tissue cysts containing bradyzoites: Present in the intermediate and definitive host; mainly in muscle and CNS
• Tissue cysts ingested by definitive host (dog) -> Oocysts produced, passed in feces, and sporulate -> Intermediate host ingests sporulated oocysts -> Zoites released and penetrates cells -> Proliferation by endodyogeny as tachyzoites -> Encyst in CNS and other tissues as bradyzoites 
• Within the intermediate host (including dogs), vertical transmission of tachyzoites can occur via transplacental transmission
  1. Transplacental (vertical) transmission is the principal route for infection in cattle
• In dogs
  1. Affected bitches are chronic carriers of the organism
  2. Pups may be subclinically infected with reactivation in later life associated with immunosuppressive illnesses, administration of modified live-virus vaccines, or glucocorticoids

 

PATHOGENESIS:

• Neospora can infect and replicate within a variety of cell types: There is affinity for cells of the monocyte-macrophage system and systemic spread occurs via leukocytic trafficking
  1. Tachyzoites have been found in neurons; reticuloendothelial cells; endothelium or tunica media smooth muscle cells; hepatocytes; muscle cells, including myocardium; and bovine placenta
  2. Tissue cysts have been found in the CNS, muscles, and retina
• Cell death is caused by active intracellular replication
• Neospora tachyzoites target the placenta in cows due to a specific lectin-binding pattern within the trophoblastic and endometrial cells (Dorsh, J Comp Pathol 2019)

 

TYPICAL CLINICAL FINDINGS:

• Young dogs infected in utero are most severely affected (horses also affected) (“postnatal syndrome”); disease is typically subclinical disease in adult dogs 
• CNS/muscular
  1. Ataxia, head tilt, hind limb paresis and rigidity that ascends, progressive paralysis. Muscle weakness, most profound in pelvic limbs. 
  2. In young dogs, clinical signs are because of an ascending polyradiculoneuritis and polymyositis
  3. In adult dogs, clinical signs are more referable to CNS lesions complicated by polymyositis, myocarditis, and dermatitis
• Recent report of a dog that presented with intractable large bowel diarrhea associated with necrosuppurative, ulcerative colitis with intra- and extracellular protozoal zoites (Curtis, J Vet Diag Invest 2020)
• Abortion: See Comp Path

 

TYPICAL GROSS FINDINGS:  

• Neuro
  1. Affecting both white and grey matter; foci of necrosis and hemorrhage in the central nervous system with vascular pattern
     • With chronicity, becomes more yellow/brown to grey and makes white matter indistinguishable from gray matter
  2. In dogs, the cerebellum is most frequently affected
  3. Can also affect spinal nerve roots and nerves, especially ventral spinal nerves
• Muscle
  1. Muscles over the entire body may be pale, streaky, and atrophic (from denervation atrophy)
  2. Marked muscle atrophy of the pelvic limb muscles occurs rapidly, and fixation of pelvic limb joints occurs as a result of denervation of muscle in an actively growing limb
• Skin: Multifocal to generalized, ulcerative papulonodular dermatitis
• Other: 
  1. Necrotic foci or granulomas, reported liver, kidneys, adrenal glands
  2. Liver may show hepatic enlargement with coalescing areas of pallor related to widespread necrosis of
  3. Lungs may show congestion and edema resulting from subacute alveolitis
  4. Necrosis and fibrinohemorrhagic enteritis
• Fetus: See Comp Path

 

TYPICAL LIGHT MICROSCOPIC FINDINGS:

• Tachyzoites: 2x6 µm crescentic organisms with small nucleus and small amounts of cytoplasm
  1. Appear in groups or clusters, either intracellular in neurons, endothelium, or epithelial cells, or extracellular
  2. May be difficult to identify on H&E
• Cysts: Round to oval, up to approximately 100 µm long, with 2-4 µm thick wall enclosing bradyzoites which are 8x2 µm
  1. Tissue cysts usually occur within CNS and less distinctly, in skeletal muscle, and less commonly within the retina
  2. There is usually little host reaction to intact tissue cysts
• Neuro
  1. Nonsuppurative encephalomyelitis: Necrotizing, granulomatous, lymphoplasmacytic, and occasionally eosinophilic meningoencephalitis with diffuse gliosis, +/- axonal swelling, digestion chamber formation
  2. Lymphohistiocytic and occasionally eosinophilic polyradiculoneuritis with secondary degeneration and necrosis in muscles or nerves
• Skin: Pyogranulomatous, occasionally eosinophilic to necrotizing and hemorrhagic dermatitis with tachyzoites in macrophages, keratinocytes, and neutrophils
• Fetus: See Comp Path
• Oocysts (passed in canine feces): Round, 10-12 µm diameter, and contain two sporocysts with four sporozoites each

 

ULTRASTRUCTURAL FINDINGS:

• Most tachyzoites located within a parasitophorous vacuole adjacent to the nucleus
• Moderately electron dense cytoplasm; single plasmalemma and double inner membrane complex; a conoid consisting of 2 microtubules arranged in a helix; 11 or more rhoptries; micronemes arranged perpendicular to the pellicle; organelles include a Golgi complex; smooth and rough endoplasmic reticulum; ribosomes; one or two mitochondria
• Tissue cyst wall 1 to 4 um thick, amorphous, non-septate

 

ADDITIONAL DIAGNOSTIC TESTS:

• Cytology
  1. 1 × 3-7 μm crescent or banana-shaped tachyzoites may be seen on cytological inspection of various fluids (abdominal effusion, bronchoalveolar lavage, cerebrospinal fluid) in infected animals 
  2. Tachyzoites of Toxoplasma gondii or Neospora caninum are not distinguishable from each other as size and morphology are very similar
• Specific diagnosis made by immunohistochemistry
• PCR, various ELISAs, indirect fluorescent antibody test (IFAT), and Neospora agglutination test (NAT)
• A competitive inhibition ELISA was developed for use in cattle (Novoa, J Vet Diag Invest 2020)
• Chromogenic in situ hybridization (ISH) is used to differentiate Toxoplasma gondii from Neospora caninum in ovine abortions with low sensitivity (Meixner, J Vet Diag Invest 2020)
• Tachyzoites are PAS positive
• Inflammatory myositis may cause an increase in creatinine kinase (CK) activity (Stockham, 2013)

 

DIFFERENTIAL DIAGNOSIS:

• Toxoplasma gondii (N-P02):
  1. Indistinguishable from Neospora by light microscopy
  2. Thin-walled cysts typically less than 1 µm thick (thinner than Neospora)
  3. Rhoptries are honeycombed; micronemes are less numerous than in Neospora
• Sarcocystis spp.:
  1. 4 x12 µm zoites
  2. Large, thin- or thick-walled cysts with septa
  3. Tachyzoites lack a parasitophorous vacuole & have a multilobed nucleus

 

COMPARATIVE PATHOLOGY:

• Dogs: See above. Also rarely causes abortion and failure of pregnancy. 
• Cattle:  
  1. In adults, the only clinical sign is abortion in dairy and beef cattle, typically at 3-9 months gestation (mid-term); fetus may be mummified, stillborn or born alive without clinical signs
  2. Lesions in fetus: Lymphocytic, plasmacytic and to a lesser extent histiocytic, hepatitis, pancarditis or myocarditis, myositis, and placentitis (no macroscopic placental lesions are present)
     • Pathognomonic CNS lesion in the fetus is the presence of multifocal discrete foci of necrosis (100-300 um in diameter) particularly in the brain and to a lesser extent in the spinal cord; in advanced lesions the necrosis can be replaced by macrophages and a few glial cells making the lesion appear as a granuloma
     • Also see multifocal pancarditis (epicarditis, myocarditis, endocarditis) and hepatitis; and protozoal organisms in either myofibers or endothelial cells
     • Lymphocytic portal hepatitis, multifocal hepatocellular necrosis, and fibrin thrombi in hepatic sinusoids are also seen
     • Foci of lymphocytes are present in additional organs, including the placenta; cotyledonary necrosis may be associated with the placentitis
  3. Difficult to recognize N. caninum tachyzoites and tissue cysts in aborted fetal brain
• Sheep and Goats: Infrequent cause of abortions and congenital infections of lambs and kids
• Horse: Rare abortions and congenital infections of foals by N. hughesi; smaller cysts with thin (<1 um thick) walls and smaller bradyzoites than N. caninum; similar to S. neurona (N-P01)
  1. Meningoencephalomyelitis, variable vasculitis and necrosis with microgliosis; perivascular cuffing with macrophages, multinucleated giant cells, lymphocytes, plasma cells or neutrophils
  2. Most common in the spinal cord gray and white matter
• NHP: Experimental infections reported in a rhesus macaques (Macaca mulatta) 
• ZEW
  1. Lesser kudu: Stillbirth with nonsuppurative encephalitis with gliosis, nonsuppurative myocarditis and tissue cysts; placental necrosis with zoites 
  2. Cervids: Reported in multiple cervids (moose, mule deer, and white-tailed deer), mostly via serology in healthy animals; rarely disease reported in young animals or fetuses 
  3. Raccoons: Has been reported secondary to canine morbillivirus infection 
  4. Rhinoceros: Infection with stillbirth documented; disseminated neosporosis with hepatomegaly, multifocal gastric ulcreration, mesenteric lymphadenomegaly and tachyzoites present in liver, adrenal cortex, kidney, and intestine reported in a white rhinoceros 

 

References:

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