JPC SYSTEMIC PATHOLOGY

HEMOLYMPHATIC SYSTEM

April 2024

H-P07 (NP)

 

SIGNALMENT (JPC# 1752289): Dog

 

HISTORY: This dog had splenomegaly and hepatomegaly.

 

HISTOPATHOLOGIC DESCRIPTION: 

SLIDE A: Spleen: Diffusely splenic red pulp is effaced by numerous macrophages, moderate numbers of plasma cells, fewer lymphocytes, and occasional neutrophils that fill and expand the cords and red pulp vascular spaces, and replace and obscure the white pulp, trabeculae, and capsule.  Macrophages are expanded up to 40µm in diameter and contain numerous 2-4 µm, ovoid, intracytoplasmic amastigotes with an eccentric, round to ovoid, basophilic nucleus and a <1 µm, linear, basophilic, kinetoplast. Multifocally, there are scattered random areas of karyorrhectic and cellular debris (lytic necrosis) admixed with numerous viable and degenerate neutrophils and variable amounts of eosinophilic, beaded to fibrillary material (fibrin). There is multifocal histiocytic erythrophagocytosis and hemosiderosis, and rare megakaryocytes (extramedullary hematopoiesis).

 

Liver: Diffusely sinusoids, portal and periportal areas, and vessels are expanded by numerous macrophages, lymphocytes, plasma cells, and fewer neutrophils. Most macrophages are filled with intracytoplasmic protozoal amastigotes as previously described. Diffusely, there is centrilobular to midzonal discontinuity, attenuation, and loss of hepatic cords. In these areas hepatocytes are rarely degenerate or necrotic, there are increased numbers of hemosiderin laden Kupfer cells, and central veins occasionally contain fibrin thrombi.

 

SLIDE B: Price Giemsa: Intracytoplasmic amastigotes have bright blue nuclei, purple kinetoplasts and light blue cytoplasm. 

 

MORPHOLOGIC DIAGNOSIS: 

1. Spleen: Splenitis, histiocytic and plasmacytic, subacute, diffuse, severe, with multifocal necrosis and numerous intrahistiocytic protozoal amastigotes, breed unspecified, canine.

 

2.  Liver: Hepatitis, histiocytic, subacute, diffuse, moderate, with multifocal hepatocellular degeneration, necrosis, and loss, and numerous intrahistiocytic protozoal amastigotes.

 

ETIOLOGIC DIAGNOSIS: Splenic and hepatic leishmaniasis

 

CAUSE: Leishmania spp. (differentials include L. infantum/chagasi, L. donovani)

 

CONDITION: Visceral leishmaniosis

 

SYNONYMS: Kala-azar, Dum dum fever

 

GENERAL DISCUSSION:  

• A zoonotic, vector-borne disease caused by an obligate intracellular (intrahistiocytic) diphasic protozoan
• Class: Kinetoplasta; Family: Trypanosomatidae
  • L. infantum (chagasi) most common; also L. donovani, L. braziliensis
• Endemic in Mediterranean countries, parts of Europe, Middle East, Africa, India, Central and South America, Texas, Oklahoma and Ohio
• Can infect a wide variety of domestic animals and wildlife; dogs common
  • Susceptible breeds: boxer, cocker spaniel, Rottweiler, German shepherd, Doberman
  • Resistant: Ibizan hound, collie breeds
• Intermediate hosts: Sandflies of the genus Phlebotomus in the Old World and the genus Lutzomyia in the New World; the tick Rhipicephalus may act as mechanical vector
  • Direct transmission and vertical transmission may occur in kenneled dogs
• Three forms of disease: Cutaneous, mucocutaneous, and visceral; dogs usually have cutaneous and visceral manifestations
  • Cutaneous leishmaniasis (Oriental sore) – The most common clinical presentation; lesions where sandfly bites (muzzle, ears, eyes); dogs may have generalized or focal cutaneous lesions
    • Eastern Hemisphere: L. tropica, L. major, L. aethiopica, L. infantum, and L. donovani  
    • Western Hemisphere: L. mexicana spp. complex (L. mexicana, L. amazonensis, L. venezuelensis) or L. braziliensis spp.

complex (L. braziliensis, L. guyanensis, L. panamensis, L.

peruviana)    

• Long standing lesions have “typical” granulomas
• Chronic proteinuric nephritis may progress to end-stage renal disease, nephrotic syndrome, and/ or systemic hypertension > leading cause of death in dogs with cutaneous infection 
• Mucocutaneous leishmaniasis (Espundia) – Spread of cutaneous infection to naso-oropharyngeal location(s); uncommon in natural animal infections
  • L. braziliensis 
  • Central America 
  • Nodular mucosal leishmaniasis affecting the oral cavity, tongue, nose and penis have been reported in the dog
• Visceral leishmaniasis (Kala-azar) – Most severe form
  • L. infantum (L. chagasi), L. donovani
  • Wide geographic distribution
  • Mimics histoplasmosis
  • Canine leishmaniasis: 
    • Used to be only sporadic in the US but is now endemic with outbreaks of L. infantum (chagasi) primarily in foxhounds in US and Canada since 1999
    • Skin lesions occur in >80% of dogs with visceral involvement
• Leishmania amastigotes have been reported in: Fibrosarcoma, T-cell lymphoma, adrenocortical adenoma, and TVT
• Other possible concomitant infections (possibly due to immunosuppression) include: Ehrlichia, Babesia, Anaplasma, Hepatozoon, Trypanosoma, Dirofilaria, Demodex, Sarcoptes, Spirocerca

 

PATHOGENESIS:   

• Sandfly ingest blood/infected host cells with amastigotes > release amastigotes in the midgut of sandfly > transform into procyclic, promastigote form and replicate > transition to infectious metacyclic form that detaches from the midgut > migrate to the mouth part > promastigotes injected into host with saliva (See below for details) 
• Female sandfly injects promastigotes (leaf shaped with a single flagellum at anterior pole) and saliva (saliva has potent vasodilating, anticoagulant, anesthetic and immunomodulatory properties) to host skin > promastigotes phagocytized by macrophages via CD35 and ICD11b/CD18 > multiply and form non-flagellated amastigotes within phagolysosomes > evasion of innate immune system via remodeling of phagosome and interference with signaling pathways > macrophages rupture (a mechanical consequence of proliferation) > freed amastigotes penetrate additional host cells and disseminate primarily through hemolymphatic system > sandfly takes a blood meal from infected host, ingests mononuclear cells containing amastigotes > transform into flagellated promastigotes 
• Severity of disease is determined by the host immune response, genetics, and concurrent disease; usually a mixed Th1/Th2 response is present in both sick and asymptomatic dogs:
  • Predominant cell-mediated immune response: Resistance to infections, TH1 dependent (IL-2, IFN-gamma, TNF-alpha)
  • Predominant humoral response: Susceptible to infection; TH2 dependent (IL-4); humoral response increases correlates with worsening of clinical signs
    • Inefficient killing with higher parasitic burden; +/- antigen:antibody complexes (type III hypersensitivity) > glomerulonephritis (+/- polyarthritis, vasculitis, uveitis); leading cause of death is chronic proteinuric nephritis (may progress to end-stage kidney, nephrotic syndrome, or systemic hypertension)
  • Autoantibody production: Polymyositis
• Recent study found that clinical status correlates with parasite load and antibody level for Leishmania infantum infection in dogs (Bruno, J Vet Diagn Invest 2019)
• Epistaxis typically associated with rhinitis, but may also be due to thrombocytopathy, increased serum viscosity due to hyperglobulinemia, or vasculitis

 

TYPICAL CLINICAL FINDINGS: 

• Visceral leishmaniasis: Mature dog in poor body condition with rough haircoat
• Other systemic signs: Fever, lethargy, cachexia, diarrhea, lymphadenomegaly, splenomegaly, hepatomegaly, weight loss
• Non-painful, non-pruritic, generalized, dry exfoliative dermatitis (most common cutaneous manifestation) with alopecia, periocular alopecia (lunettes), recurrent oculonasal discharge, nasal crusting, epistaxis, +/- concurrent conjunctivitis, uveitis, and blepharitis, onychogryphosis 
• Most common laboratory finding is hyperproteinemia characterized by hypergammaglobulinemia and hypoalbuminemia (due to plasma cell hyperplasia); also proteinuria, azotemia, elevated ALP and ALT, mild non-regenerative anemia, lymphopenia, thrombocytopenia
• Skin lesions occur in over 80% of dogs with visceral involvement
• Dogs occasionally present with only ulcerative nodular oral lesions (Blume, J Comp Pathol 2019)
• Dogs may present with chronic colitis; 7% of cases of chronic colitis from endemic areas were immunohistochemically positive for Leishmania (Casanova, J Comp Pathol 2019)

 

TYPICAL GROSS FINDINGS:

• Visceral: Generalized lymphadenopathy, hepatomegaly, splenomegaly, nodular granulomas (liver, kidney); erosion/ulceration/crusting (oral, nasal, GI mucosa, mucocutaneous junction, skin, synvoiae); soft red bone marrow
• Cutaneous: Alopecia, ulcerative or exfoliative dermatitis, nasodigital hyperkeratosis, onychogryphosis
  • Dogs with generalized and focal lesions; most severe on muzzle, periorbital (“periocular lunettes”), and aural regions (where sandflies feed); nodular mucosal leishmaniasis is also reported
    • Ulcerative, multinodular dermatitis > mucocutaneous proliferative dermatitis and papular dermatitis  
    • Ulcerations over bony prominences and mucocutaneous junctions > sequelae of inflammation extending to the overlying epithelium  
• Less common: Masticatory myositis, myositis of other muscles, anterior uveitis, keratoconjunctivitis sicca, epistaxis (uni- or bilateral), non-erosive polyarthritis, polymyositis, osteolysis/osteomyelitis, osteoarthritis, proliferative periostitis, glossitis/stomatitis, pancreatitis, chronic colitis, myocarditis, meningitis, meningoencephalomyelitis, orchitis/epididymitis/prostatitis, myocarditis; Leishmania infantum is a reported cause of failure of pregnancy in dogs

 

TYPICAL LIGHT MICROSCOPIC FINDINGS:  

• Organism: Intrahistiocytic amastigotes: ovoid to round, 2-5 µm diameter, with a clear halo, a round, vesicular basophilic nucleus with rod-like dark staining kinetoplast adjacent nucleus (no flagellum); occasionally within other leukocytes, endothelial cells, fibroblasts or neoplastic cells
• Spleen: 
  1. Initial disease- Hemic-lymphatic hypertrophy with macrophage proliferation and focal granulomas, hyperplastic splenic follicles, often with follicular hyalinosis
  2. Advanced disease- Atrophy of lymphoid follicles, sinus areas expanded by large macrophages heavily laden with intracytoplasmic organisms, and numerous plasma cells
• Bone marrow: Clusters of epithelioid macrophages with phagocytosed organisms, plasma cell hyperplasia (up to 50%)
• Liver: Granulomas
• Skin: Nine inflammatory patterns of leishmaniasis; 3 most common are granulomatous to lymphoplasmacytic perifolliculitis, superficial and deep perivascular dermatitis, and interstitial dermatitis
• Kidney: Membranoproliferative glomerulonephritis; variable interstitial scarring with some parasitic involvement, amyloid may be present
• Heart: Lymphoplasmacytic or granulomatous myocarditis (especially right atrial); myocardial necrosis and increased interstitial collagen, are prevalent in dogs
• Eye: Ocular inflammation: Conjunctiva, limbus, ciliary body, iris, cornea, sclera/iridocorneal angle, choroid, optic nerve sheath
• CNS: Intramedullary spinal cord mass, meningoencephalomyelitis, vasculitis 
• Bone marrow: +/- focal lesions: Epithelioid macrophages with phagocytized organisms

 

ULTRASTRUCTURAL FINDINGS:  

 

ADDITIONAL DIAGNOSTIC TESTS:  

• Cytology, Giemsa, immunohistochemistry, in situ hybridization, PCR, serology (IFA, ELISA)
• On cytology: Macrophages predominate, with intracellular amastigotes with a red nucleus and characteristic bar-shaped kinetoplast
• Blood smears: Rare detection of intraneutrophilic and intramonocytic Leishmania amastigotes (Oikonomidis, J Vet Diagn Invest 2019)
• ANA and Rheumatoid Factor are weak or absent in cases of arthritis (Tsouloufi, J Vet Diagn Invest 2022)

 

DIFFERENTIAL DIAGNOSIS: 

• Visceral disease mimics histoplasmosis
• Gross: Conditions causing hyperkeratosis of the nasal planum or pawpads in dogs
  1. Sarcoptic/demodectic mange, seborrhea, pemphigus foliaceus, SLE, bacterial infection, superficial necrolytic dermatitis, Zn-responsive dermatitis, neoplasia (cutaneous lymphoma)

 

Microscopic:

• Trypanosoma cruzi (tissue phase/amastigote form): Most often found within cardiomyocytes
• Histoplasma capsulatum: 2-5µm, intrahistiocytic, narrow-based budding; predominantly histiocytic inflammation; visceral leishmaniasis mimics histoplasmosis
• Toxoplasma gondii: 2-6µm tachyzoites; necrosis
• Neospora caninum: 4-7µm tachyzoites
• Sporothrix schenckii: 4-10µm, oval to cigar shaped yeast
• Neorickettsia helminthoeca: Intracellular; gram negative; organisms are 0.3-2.0 µm, pleomorphic cocci or rods 

 

COMPARATIVE PATHOLOGY:  

• Cats: L. Mexicana (endemic in Texas) has cutaneous tropism; only 5 of 8 cases in this report stained with Giemsa; pinnae most frequent site of nodule formation 
• Captive bush dogs, crab-eating foxes, golden jackals, hoary foxes, and maned wolves: Cutaneous and visceral leishmaniasis
• Free-ranging genets: Leishmania infantum infection reported, but disease and associated clinical signs not described
• Captive red and common northern kangaroos: Leishmania sp. reported 
• Black wallaroos and agile wallabies: Cutaneous leishmaniasis reported; lesions on the tail, inner forearms, and hind legs 
• Hyraxes: Emerging disease of concern due to zoonotic transmission of L. tropica and L. aeithopica, potential reservoir host and origin of human infection in Ethiopia and Middle East, does not commonly causes disease in rock hyraxes
• Bats, in Brazil:
  • Free-ranging Artibeus lituratus: Leishmania (Leishmania) mexicana reported in dead animal with raised, ulcerated mass on the leading edge of the patagium 
  • Glossophaga soricina: Cutaneous lesions associated with L. (Viannia) braziliensis in two animals
  • L. (Leishmania) amazonensis and L. (Leishmania) infantum chagasi detected 
• Agouti: Leishmania lainsoni reported in these rodents
• Horse: Cutaneous Leishmania martiniquensis on pinnae in Florida (Menezes J Comp Path 2019)

 

REFERENCES:  

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3. Blume GR, Eloi RSA, Silva FP, Eckstein C, Santos RL, Sant'Ana FJF. Oral Lesions in Dogs with Visceral Leishmaniosis. J Comp Pathol. 2019;171:6-11.
4. Bruno B, Romano A, Zanatta R, et. al. Serum indirect immunofluorescence assay and real-time PCR results in dogs affected by Leishmania infantum: evaluation before and after treatment at different clinical stages. J Vet Diagn Invest. 2019; 31:(2)222-227.
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