JPC SYSTEMIC PATHOLOGY

HEMOLYMPHATIC SYSTEM

February 2024

H-B11 (NP)

 

Signalment (AFIP # POLA 27): German shepherd dog, age and gender not specified.

 

HISTORY: A blood smear was obtained from an emaciated German shepherd dog.

 

HISTOPATHOLOGIC DESCRIPTION: Peripheral blood smear: The sample is of low cellularity. Rarely, monocytes contain round to oval, 4-6 µm diameter, basophilic intracytoplasmic rickettsial morulae.  

 

MORPHOLOGIC DIAGNOSIS: Cytologic specimen, peripheral blood smear:  Intramonocytic ehrlichial morulae, few, German shepherd dog, canine.

 

CAUSE: Ehrlichia canis

 

CONDITION: Canine monocytotropic ehrlichiosis (CME)

 

SYNONYMS: Canine tropical pancytopenia, tracker dog disease, canine hemorrhagic fever

 

GENERAL DISCUSSION:

• Ehrlichia canis, a small, pleomorphic, gram-negative, rickettsial organism that causes canine monocytotropic ehrlichiosis, infecting leukocytes
• Transmitted by the brown dog tick (Rhipicephalus sanguineus)
• Causes infection in dogs and rarely cats worldwide; German shepherd dogs are very susceptible 

 

PATHOGENESIS:

• Infection occurs when an infected tick ingests a blood meal and salivary secretions contaminate the feeding site
• Ehrlichia canis survives in membrane-bound vesicles within lymphocytes and monocytes
• Thrombocytopenia with reduced platelet adhesion and aggregation occurs due to an unknown mechanism 
• Experimental infection induces minimal change disease within kidneys; severe proteinuria with reversible global fusion of podocyte foot processes ultrastructurally, but glomeruli histologically normal

 

TYPICAL CLINICAL FINDINGS:

• There are three phases of infection – Acute, subclinical, and chronic

1. Acute phase – Fever, leukopenia, moderate to severe thrombocytopenia, anemia, edema, petechia, icterus, ataxia, reluctance to move (lameness), lymphadenopathy, and/or splenomegaly; ophthalmic lesions (anterior uveitis, chorioretinitis, papilledema, retinal hemorrhage); morulae observed on blood smears
2. Chronic phase – Severely ill, marked weight loss, marked lymphocytosis (a non-neoplastic immune response with large granular lymphocytes or small, mature lymphocytes) or severe pancytopenia 

 

CLINICAL PATHOLOGY:

• Most commonly, aplastic anemia with pancytopenia (acute) or lymphocytosis (chronic) 
• Morulae on blood smears or synovial fluid – Round to oval, pink to blue membrane bound vesicles containing clusters of minute cocci in cytoplasm of lymphocytes and monocytes; rarely seen on blood smears except in very early stages of infection
• Proteinuria 

 

TYPICAL GROSS FINDINGS:

• Splenomegaly, lymphadenomegaly, pulmonary edema, hemorrhage 
• +/- multisystemic petechiae, ecchymoses
• Widespread petechial and ecchymotic hemorrhages on serosal and mucosal surfaces of most organs, and on gingiva and conjunctiva; large subcutaneous hemorrhages
• Lungs: Mottled and edematous; focal hemorrhages
• Acute phase: Generalized lymphadenomegaly, splenomegaly, hepatomegaly, red bone marrow
• Chronic phase: Loss of body condition, emaciation, subcutaneous and interstitial edema, pale bone marrow

 

TYPICAL LIGHT MICROSCOPIC FINDINGS:

• Morulae of E. canis (difficult to identify on histology): Round, basophilic structures 1/3 to 1/2 the diameter of erythrocytes, with a granular internal basophilic elementary bodies (0.5-1.0 µm in diameter); within mononuclear leukocytes
• Prominent perivascular plasmacytic infiltrate and vasculitis in many organs, including multifocal nonsuppurative meningoencephalitis 
• Kidney – Amyloidosis 
• Bone marrow hypoplasia with chronicity

 

ADDITIONAL DIAGNOSTIC TESTS:

• IFA assays (e.g. SNAP 4Dx) – Do not completely differentiate between richettsial organisms and may have false-negative results if titers are low 
• PCR - Most reliable to differentiate between Ehrlichia and Anaplasma species 
• CSF analysis – Neutrophilic pleocytosis 
• PARR – Can have a clonal receptor gene rearrangement of the T-cell receptor (mimicking lymphoma)

 

DIFFERENTIAL DIAGNOSIS: 

• Anaplasma phagocytophilum (formerly E. equi, E. phagocytophila, Cytoecetes phagocytophila, Rickettsia phagocytophila) – Obligate intracellular, gram-negative bacterium transmitted by ixodid ticks (Ixodes scapularis); infects and survives in morulae within neutrophils; acute infection identical to E. canis, except ophthalmic lesions are uncommon; if immunocompetent, infection is self-limiting due to a robust immune response; diagnosed via morulae on blood smears, antibody titers, and PCR; has been linked to canine granulocytic anaplasmosis and canine cyclic thrombocytopenia 
• Rocky Mountain spotted fever (Rickettsia rickettsii): Febrile exanthema of primarily <2 year-old dogs, especially German shepherd dogs; acute infection identical to E. canis; transmittd by Dermacentor variabilis and D. andersoni; public health risk as humans can be infected while removing ticks from dogs; rickettsial organism invades and replicates within endothelial cells of small vessels > endothelial damage and vasculitis > platelet activation and coagulation cascade activation > thrombosis; clinically, dogs are listless, depressed, febrile, anemia, leukopenic, thrombocytopenic, hyperfibrinogenemic; endothelial damage due to phospholipase A and trypsin-like protease of rickettsial or host immune system origin and thrombocytopenia due to antiplatelet antibodies; gross lesions include edema, dermatitis, petechia and ulceration of mucous membranes, abdominal skin, pleura, and gastric wall, hemorrhagic colitis, and hemorrhagic lymphadenopathy; death may occur in acute disease due to peripheral vascular collapse, hemorrhagic diathesis, thrombosis or rarely disseminated intravascular hemolysis; prominent histologic lesion is necrotizing vasculitis of small veins, capillaries, and arterioles with perivascular monocytic cuffing
• Do not confuse morulae with Dohle inclusion bodies, platelets lying on leukocytes or blebs of nuclear membrane 

 

COMPARATIVE PATHOLOGY:

Rickettsia that infect ENDOTHELIUM: Rickettsia rickettsia – Rocky Mountain spotted fever (see above) 

Rickettsia that infect ERYTHROCYTES:

• Anaplasma marginale: Causes bovine anaplasmosis; world wide distribution; marked regenerative anemia via immune-mediated extravascular hemolysis, primarily in cattle >3 years old (calves resistant, as are indian cattle [Bos indicus]); often have jaundice and bilirubinemia/uria, but no hemoglobinemia/uria (no intravascular hemolysis unlike babesiosis); obligate intracellular bacteria located in membrane-bound (initial) bodies at erythrocyte margin; transmitted biologically by ixodid ticks (Dermacentor, Rhipicephalus) or mechanically by flies, mosquitos, or blood contaminated fomites; identification of organisms diagnostic, but difficult to find in chronically infected carrier animals; ELISA or PCR useful; surviving animals become carriers (reservoirs); no pathognomic gross lesions of anaplasmosis, but results in mucous membrane pallor (anemia), icterus, splenomegaly, and hepatomegaly 
• Anaplasma centrale: Another cause of bovine anaplasmosis, but rarely causes disease; obligate intracellular bacteria located in center of erythrocyte; mostly restricted to South America, Africa and the Middle East 
• Anaplasma ovis: Ovine and caprine anaplasmosis; rarely causes overt disease unless animal has concurrent infections; disease is more common in goats
• Anaplasma phagocytophilum and Ehrlichia ruminatium may also infect endothelium 

Rickettsia that infect LEUKOCYTES:

• Preferentially infects granulocytes: 
  • Ehrlichia ewingii: Canine granulocytic ehrlichiosis; only present in USA; organisms detectable in neutrophils in blood smears or synovial fluid; transmitted by Ambylomma americanum
  • Anaplasma phagocytophilum (formerly E. equi, E. phagocytophila, Cytoecetes phagocytophila, Rickettsia phagocytophila): Causes disease in ruminants (tick-borne fever), horses (equine granulocytic anaplasmosis), dogs (see above), and cats; sudden onset of fever and may abort if pregnant; with onset of fever, organisms detectable in circulating neutrophils and large lymphocytes with Giemsa or methylene blue 
• Preferentially infects mononuclear cells: 
  • Ehrlichia canis: See above
  • Ehrlichia chaffeensis: Canine monocytic ehrlichiosis; transmission by lone star tick (Ambylomma americanum) and reservoir is white-tailed deer; outbreaks also reported in captive lemurs with thrombocytopenia and leukopenia 
• Infects both granulocytes and mononuclear cells: 
  • Ehrlichia ruminantium (formerly Cowdria ruminantium): Causes heartwater (cowdriosis) in cattle, sheep, goats, and other exotic ruminants in Africa and on a few islands in the Caribbean; vector-borne (Amblyomma ticks); characteristic pericardial effusion in small ruminants (unknown pathogenesis), as well as other cavitary effusions, edema, and splenomegally; cross reacts with A. phagocytophilum and E. canis with IFA; calves and lambs <3 weeks old are resistant; young adults have 60% (cattle) to 100% (European sheep) mortality; those that survive develop immunity of variable duration; initially infects reticulum cells and macrophages of lymph nodes and spleen (forming initial bodies), but then endothelium (forming characteristic morulae in the cytoplasm at the poles of endothelial nuclei, which ultrastructurally can be classified as elementary [electron dense], intermediate, or reticulate bodies); peracute disease results in convulsions and rapid death; acute disease (most common) lasts up to 6 days with high fever, dyspnea, neurologic signs, diarrhea (cattle), anemia, leukopenia and hypoalbuminemia; subacute disease is prolonged; diagnose with cytologic smears from brain or large vessels, PCR, IFA, ELISA, MAP

Rickettsia that primarily infect PLATELETS: Anaplasma platys (formerly Erhlichia platys)- Infectious canine cyclic thrombocytopenia; transmitted by Rhipicephalus sanguineus; has also been linked to canine granulocytic anaplasmosis 

Rickettsia that primarily cause GASTROINTESTINAL DISEASE:

• Neorickettsia risticii (formerly E. risticii):  Equine monocytic ehrlichiosis or Potomac horse fever; fever, colitis
• Neorickettsia helminothoeca: Salmon poisoning in dogs; lymphadenitis, diarrhea 

 

REFERENCES

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2. Cianciolo RE, Mohr FC. Urinary system. In: Maxie MG, ed. Jubb, Kennedy, and Palmer’s Pathology of Domestic Animals. Vol 2. 6th ed. St. Louis, MO: Elsevier Limited; 2016:414, 418.
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4. Fernandes PJ. Synovial Fluid Analysis. In: Valenciano AC, Cowell RL, eds. Diagnostic Cytology and Hematology of the Dog and Cat. 5th ed. St. Louis, MO: Elsevier Mosby; 2014:197, 199. 
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8. Ramos-Vara JA, Gelain ME. Chapter 18: Advanced Diagnostic Techniques. In: Raskin RE, Meyer DJ, & Boes KM eds. Canine and Feline Cytopathology: A Color Atlas and Interpretation Guide. 4th ed. St. Louis, MO: Elsevier; 2022:653.
9. Richardson SS, Mainville CA, Arguello-Marin A, et al. A second-generation, point-of-care immunoassay provided improved detection of Anaplasma and Ehrlichia antibodies in PCR-positive dogs naturally infected in Anaplasma or Ehrlichia species. J Vet Diagn Invest. 2023;35(4):366-373. 
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