JPC SYSTEMIC PATHOLOGY
MUSCULOSKELETAL SYSTEM
APRIL 2022
M-P01

Signalment (JPC #2209517):  Dog

 

HISTORY:  This dog had muscle weakness.

 

HISTOPATHOLOGIC DESCRIPTION:  Skeletal muscle: Multifocally effacing and replacing myofibers are discrete, 175um diameter aggregates of neutrophils, macrophages, and fewer lymphocytes and plasma cells (pyogranulomas) with frequent intrahistiocytic, 3-4um, spherical protozoal merozoites.  Multifocally separating and compressing myocytes are multilamellar mucopolysaccharide cysts (“onion skin cysts”) that are up to 200 um diameter composed of concentric lamellations of amphophilic, mucinous material further surrounded by 1-3 layers of spindle cells, all centered on a macrophage with abundant foamy to granular cytoplasm, a single nucleus with a prominent nucleolus, and occasionally an intracytoplasmic, round to oval,  6-10um, eosinophilic protozoal trophozoite. Rarely within the center of the cyst, there is a 75um meront containing numerous 4-5 um diameter merozoites. Diffusely there is marked loss of myofibers; remaining myofibers are either shrunken (atrophy), have pale, swollen, vacuolated sarcoplasm (degeneration), have hypereosinophilic, fragmented sarcoplasm with loss of cross striations and nuclear pyknosis or karyolysis (necrosis) and occasional granular basophilic sarcoplasm (mineralization), have increased sarcoplasmic basophilia with multiple central nuclei (regeneration), or are rarely multinucleated, misshapen, regenerative myocytes (muscle giant cells, abortive regeneration). The perimysium and endomysium are diffusely expanded up to 50 um by abundant fibrosis and edema with few scattered macrophages, neutrophils, lymphocytes, and plasma cells.

MORPHOLOGIC DIAGNOSIS:  Skeletal muscle:  Myositis, pyogranulomatous, diffuse, chronic, marked, with myocyte degeneration, necrosis, regeneration, and atrophy, fibrosis, and numerous mucopolysaccharide (onion skin) protozoal cysts and merozoites, breed unspecified, canine.

 

ETIOLOGIC DIAGNOSIS:  Hepatozoon myositis

 

CAUSE:  Hepatozoon americanum

 

CONDITION:  American canine hepatozoonosis

 

GENERAL DISCUSSION:

• Hepatozoon americanum is a tick-borne apicomplexan protozoan (family Hemogregarinidae)
• First recognized in the U.S. in 1978 on the Gulf of Mexico coast; this organism is now diagnosed in dogs throughout much of the southeastern U.S and is spreading to other states
• Clinical signs, laboratory abnormalities, pathological lesions, tissue tropism, parasite morphology, and tick vectors differ from disease associated with canis
• Prominent features of disease: Skeletal and cardiac myositis; disseminated periosteal bone proliferation; massive neutrophilia
• Parasitized cells are of leukocyte origin; when in muscle, parasites are in macrophages between muscle fibers and are induced to produce mucopolysaccaride which forms “onion skin” lamellations

 

PATHOGENESIS

• Transmitted via ingestion of the definitive host (Amblyomma maculatum); predation with ingestion of cystozoites from infected host tissues; NOT transmitted via tick bite
• americanum can cause debilitating disease even without concurrent immunosuppression (unlike H. canis)
• Cysts can lie dormant for long periods of time; cycles of asexual reproduction continue when activated (waxing and waning of disease)
• Pyogranulomatous myositis in muscles may stimulate marked periosteal proliferation (especially in younger animals), generally of proximal long bones first
• Chronic infection > persisitent antigenic stimulation > vasculitis, proliferative glomerulonephritis, amyloid deposition, uveitis

 

LIFE CYCLE

• Two hosts required for life cycle:
  • Definitive host: Gulf Coast tick (Amblyomma maculatum)
  • Intermediate host: Dogs and other canids
• Immature ticks feed on infected intermediate host > ingest leukocytes containing gamonts > sexual reproduction in tick > oocysts (contain numerous sporocysts, each with 10-26 sporozoites) > tick molts into adult (mature oocysts in hemocoele) > dog ingests infected tick > sporozoites penetrate intestinal wall > enter circulation and move to muscle and target tissues > parasite develops in macrophages between myocytes > layers of mucopolysaccharides laid down around host cell, forming "onion-skin" cyst > asexual reproduction > when mature, cyst ruptures > merozoites released > severe inflammatory response (neutrophils and monocytes) > infected inflammatory cells return to circulation > spread to distant sites or become gamonts > ingested by ticks

 

TYPICAL CLINICAL FINDINGS

• H. americanum causes a debilitating and usually fatal disease; chronic infection may have waxing and waning or progressively worsening clinical signs
• Clinical signs due to myositis and periosteal bone formation; may mimic those of meningitis or discospondylitis; gait abnormalities; pain; weight loss despite a good appetite; muscle wasting (may be severe); depression; mucopurulent ocular discharge; pyrexia (up to 106 degrees F)
• Clinical pathology
  • CBC:
    • Leukocytosis (may be severe or leukemoid response)– ranges from 20,000 to 200,000; characterized by mature neutrophilia +/- left shift)
    • Anemia: Mild to moderate, normocytic, normochromic, nonregenerative
    • Platelet count: Normal to elevated
    • Persistent mild eosinophilia
• Serum Chemistry:
  • Glucose: Artifactually decreased because of increased metabolism of glucose by high numbers of leukocytes (not true hypoglycemia)
  • Albumin: Decreased (decreased intake, chronic inflammation, renal loss) with corresponding hypocalcemia
  • Globulins: Elevated
  • ALP: Mild elevation (due to periosteal inflammation)
  • BUN: Decreased (reduced protein intake, negative nitrogen balance, production of inflammatory proteins)
  • Low glucose, albumin, and BUN with elevated ALP may be misinterpreted as liver failure; bile acids are normal or slightly elevated
  • CK: Almost always normal despite myositis
• Urinalysis: Some infected dogs may develop immune-mediated glomerulonephritis with proteinuria and elevated urine protein:creatinine ratio

 

TYPICAL GROSS FINDINGS

• Cachexia, muscular atrophy
• Disseminated, symmetric, periosteal new bone formation that most frequently and most severely involves the diaphysis of long bones (femur, humerus, radius, ulna), vertebral column, and pelvis
• Pyogranulomas: 1-2 mm diameter white/tan foci scattered throughout muscle and other tissue

 

TYPICAL LIGHT MICROSCOPIC FINDINGS

• Muscle biopsy: Identification of cysts, meronts, and pyogranulomas with zoite-containing leukocytes (macrophage) is diagnostic
• Myositis with muscle atrophy, necrosis, inflammatory cell infiltrates
• "Onion-skin cysts" in skeletal and cardiac muscle: Round to oval with single round basophilic nucleus surrounded by concentric layers of light blue staining laminar membranes (nonsulfated acid mucopolysaccharide); 250 to 500 um in diameter; may have developing meront inside
  • Cysts have also been found in intestinal smooth muscle, adipose tissue, pancreas, spleen, lymph node, liver, skin, kidneys, lungs
• Pyogranulomas
• Bone: Periosteal reaction primarily of the more proximal diaphyseal regions, vertebrae, and ilium – woven bone forming trabeculae perpendicular to the cortex
• Kidneys: Renal lesions common; focal pyogranulomatous inflammation with mild glomerulonephritis; lymphoplasmacytic interstitial nephritis; mesangioproliferative glomerulonephritis
• Chronic infections: Amyloid deposits in spleen, lymph nodes, small intestine, liver, kidneys; uveitis
• Vascular changes include fibrinoid degeneration of vessel walls, mineralization and proliferation of tunica media, and pyogranulomatous vasculitis

ULTRASTRUCTURE

• Apicomplexan protozoa: Conoid, rhoptries, micronemes, micropore, pellicle with subpellicular microtubules
• Unique to Hemogregarinidae: Rhoptries both anterior and posterior to nucleus

 

ADDITIONAL DIAGNOSTIC TESTS

• Blood smears: Unreliable; gamonts/gametocytes (oval to elliptical, pale blue, 9 x 4um) are rarely observed within neutrophils, may peripheralize the nucleus
• Serology: ELISA:  highly sensitive (93%) and specific (96%) when compared to muscle biopsy
• IFA, PCR, IHC
• Bone marrow aspirates: Increased M:E ratio (granulocytic hyperplasia); usually do not see organisms
• Lymph node aspirates: Lymphoid hyperplasia; no organisms

 

DIFFERENTIAL DIAGNOSIS:

• Other protozoan parasites:
  • Sarcocystosis, Toxoplasma gondii, Neospora caninum: Muscle lesions develop from cysts with a myriad of zoites but no “onion-skin cysts”
  • Hepatozoon canis: Carnivorous mammals in Africa, southern Europe, Asia (including Middle East), islands of Pacific and Indian Oceans
    • No muscle lesions; form classic “spoke wheel” schizonts in hemapoietic tissue
    • Infects hemolymphatic tissues and can cause a mild anemia and lethargy
    • Neutrophil is the favored host cell and zoites can be seen histologically
    • Rhipicephalus sanguineus (brown dog tick) is the invertebrate host
  • Trypanosoma cruzi: pseudocysts with amastigotes containing a parallel kinetoplast, in cardiac muscle, no “onion-skin cysts”
• Other causes of periosteal proliferation:
  • Hypertrophic osteopathy: Very similar histologically and grossly

 

COMPARATIVE PATHOLOGY:

Hepatozoon spp. in other veterinary species:

• Rodents: H. muris, H. balfouri
• Gray squirrels: H. griseiciuri
• Raccoons: H. procyonis; common in Texas Gulf Coast region
• Kiwi: H. kiwii, gametocytes are present within monocytes, Ixodes anatis (kiwi tick) is thought to be the vector, but this has not been verified
• Bats: Hepatozoon spp have been reported in free ranging bats, no associated clinical disease
• H. americanum or a similar organism has also been described in coyotes, bobcats, and ocelots in the southeastern U.S.

 

REFERENCES:

1. Baneth G. Perspectives on canine and feline hepatozoonosis. Vet Parasitol. 2011;181:3-11.
2. Cooper BJ, Valentine BA. Muscle and tendon. In: Maxie MG, ed. Jubb, Kennedy and Palmer’s Pathology of Domestic Animals. Vol 1. 6th ed. Louis, MO: Elsevier; 2016:240.
3. Craig LE, Dittmer KE, Thompson KG. Bones and joints. In: Maxie MG, ed. Jubb, Kennedy and Palmer’s Pathology of Domestic Animals. Vol 1. 6th ed. Louis, MO: Elsevier; 2016:94,98.
4. Cummings CA, Panciera RJ, Kocan KM, Mathew JS, Ewing SA. Characterization of stages of Hepatozoon americanum and parasitized canine host cells. Vet Pathol. 2005;42(6):788-796.
5. Farina LF, Lankton JS. Chiroptera In: Terio K, McAloose D, St. Leger J, eds. Pathology of Wildlife and Zoo Animals, San Diego, CA: Elsevier 2018: 626.
6. Keel, MK, Terio, KA, McAloose, D. Canidae, Ursidae, Ailuridae In: Terio K, McAloose D, St. Leger J, eds. Pathology of Wildlife and Zoo Animals, San Diego, CA: Elsevier 2018: 250.
7. Macintire DK, Vincent-Johnson NA, Potter M. Hepatozoon americanum In: Greene C, ed. Infectious Diseases of the Dog and Cat. 4th ed. St Louis MO: Elsevier Saunders; 2012: 757-763.
8. Parkins, ND, Stokes JV, Gavron NA, Frankovich AN, Varela-Stokes, JV. Scarcity of Hepatozoon americanum in Gulf Coast tick vectors and potential for cultivating the protozoan. Vet Parasitol. 2020; 21.
9. Smith DA. Palaeognathae: Apterygiformes, Casuariiformes, Rheiformes, Struthioniformes; Tinamiformes In: Terio K, McAloose D, St. Leger J, eds. Pathology of Wildlife and Zoo Animals, San Diego, CA: Elsevier 2018: 647.
10. Stockham SL, Scott MA. Fundamentals of Veterinary Clinical Pathology. 2nd ed, Ames, Iowa: Blackwell 2008: 74, 86, 95-97.
11. Valli VEO, Kiupel M, Bienzle D. Hematopoietic system. In: Maxie MG, Jubb, Kennedy and Palmer’s Pathology of Domestic Animals. Vol 1. 6th ed. Philadelphia, PA: Elsevier; 2016:110-111.

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