JPC SYSTEMIC PATHOLOGY

INTEGUMENTARY SYSTEM

October 2022

I-P15

 

Signalment (JPC# 2277382): Two-year-old male boxer

 

HISTORY: This dog was underweight and had hyperkeratosis of the planum nasale, ulceration of the oral mucocutaneous junction, and erythematous and edematous lesions on the feet.

 

HISTOPATHOLOGIC DESCRIPTION

Slide A: Haired skin and subcutis: Diffusely infiltrating the dermis, extending to and elevating the mildly hyperkeratotic epidermis, surrounding, separating, and replacing collagen bundles and adnexa, and multifocally extending into the subcutis are numerous macrophages as well as plasma cells, and fewer lymphocytes and eosinophils, and occasional multinucleated giant cells (Langhans type). Macrophages often contain protozoa that are occasionally within a clear parasitophorous vacuole or, to a lesser extent, free within the extracellular space. Protozoa are 2-3µm in diameter with clear cytoplasm and a single 1µm basophilic nucleus. The superficial dermis is multifocally mildly expanded by edema and fibrin, and dermal collagen is multifocally smudged. There is multifocal minimal orthokeratotic hyperkeratosis There is moderate fibrosis within the deep dermis and subcutis. Myocytes within the panniculus carnosus are often either shrunken (atrophy), swollen with vacuolated sarcoplasm (degeneration), or shrunken with hypereosinophilic sarcoplasm and pyknotic nuclei (necrosis).

 

Slide B: Haired skin and subcutis (Giemsa): There are numerous intrahistiocytic and fewer extracellular 2-3µm diameter, deeply-purple amastigotes with clear cytoplasm and a 1µm diameter nucleus with a smaller adjacent, often perpendicular kinetoplast.

 

MORPHOLOGIC DIAGNOSIS: Haired skin and subcutis, site not specified: Dermatitis and panniculitis, granulomatous and plasmacytic, multifocal to coalescing, marked, with numerous intrahistiocytic and fewer extracellular amastigotes, boxer, canine.

 

ETIOLOGY: Leishmania spp.

 

ETIOLOGIC DIAGNOSIS: Cutaneous leishmaniasis

 

GENERAL

• Leishmania is an important cause of cutaneous and visceral disease in a number of species including canids and humans; can manifest as a subclinical infection, self-limiting disease, or severe systemic illness
  1. Visceral leishmaniasis: H-P07 (spleen, liver) and U-P03 (kidney)
• Class: Kinetoplasta; Family: Trypanosomatidae; many species, L. infantum (syn L. chagasi) common
  1. Other spp. include L. donovani, L. braziliensis, and L. Mexicana
  2. Subgenera exist within Genus Leismania, including Leishmania and Mundinia
     • E.g. Leishmania (Mundinia) martiniquensis
• Obligate intracellular protozoa that infect monocytes and macrophages, and are transmitted to mammalian reservoir hosts (typically canids) by sandflies (Phelbotomus in the Old World; Lutzomyia in the New World)
• Endemic to many parts of the world, but disease mainly occurs in the Mediterranean basin, Asia and South America
  1. Also found in parts of Africa, Central America; certain species are endemic to the U.S. with cases occurring without travel history 
  2. Leishmaniasis is also endemic in Foxhound populations in the United States
• Dogs are natural hosts and the primary domestic reservoir for human infection
• Three forms of the disease: Cutaneous, mucocutaneous, and visceral; dogs usually only have cutaneous and visceral manifestations (mimics histoplasmosis)

 

PATHOGENESIS AND LIFE CYCLE

• Lifecycle:
  1. Phelbotomus or Lutzomyia sandfly (intermediate host) ingests cells infected with amastigotes from infected host ->
  2. In sandfly midgut, amastigotes released from cells, transform into flagellated, procyclic, promastigote form and replicates via binary fission ->
  3. Promastigotes transition into infectious metacyclic form -> migrate to sandfly mouthpart -> 
  4. Promastigotes regurgitated during sandfly blood feeding -> promastigotes phagocytized by host macrophage -> multiply as non-flagellated amastigotes within phagolysosome -> 
  5. Amastigotes proliferate and proliferate (binary fission) -> rupture out of macrophages -> infect new cells
     • Can disseminate through hemolymphatic system
• Transmission:
  1. Bite of a female sandfly (Phlebotomous spp., Lutzomia spp.) 
  2. Mechanical vectors (e.g. Rhipicephalus tick or fleas)
  3. Transfer of blood or body fluids (e.g. blood transfusion)
  4. Venereal transmission from infected males to healthy bitches documented
  5. Vertical, in utero transmission
• Incubation period may vary from weeks to years
• Innate immunity evaded by organism; adaptive (cell-mediated) immunity plays major role in host defense
  1. Infection resistance associated with Th1-type immune response; susceptibility associated with Th2-type immune response
     • Protective immunity associated with cytokines (e.g., TNF-β, IL-2, and IFN-γ) released by activated T lymphocyte that upregulate macrophage killing of the organisms and by CD8+ cytotoxic T lymphocytes killing infected macrophages
  2. Clinically ill dogs have decreased T-lymphocyte-mediated immunity and increased B-lymphocyte proliferation
     • Can lead to hyperglobulinemia and disease (see below)
  3. Clinical and histologic [cutaneous] lesions may be useful in establishing a prognosis. E.g.:
     • Alopecic form in the dog: Associated with fewer organisms and a more appropriate cellular immune (number of antigen-presenting Langerhans cells, major histocompatibility complex II–positive keratinocytes, and infiltrating T cells)
     • Nodular form in the dog: Lack antigen-presenting cells, more numerous macrophages containing large numbers of organisms
     • Papular dermatitis in the dog: Associated with good response to treatment, suggestive of immunocompetence and favorable prognosis
• Disease can be from a variety of pathogenic mechanisms:
  1. Granulomatous inflammation (e.g. nodular dermatitis, osteomyelitis)
  2. Immune-complex deposition (e.g. glomerulonephritis, interstitial nephritis)
  3. Autoantibody production (e.g. polymyositis)
• The leading cause of death in affected dogs is immune complex deposition in glomeruli, which causes chronic protein-losing nephritis (nephrotic syndrome) that may progress to end-stage kidney disease, nephrotic syndrome, and/or systemic hypertension

 

TYPICAL CLINICAL FINDINGS

• Cutaneous leishmaniasis:
  1. Alopecia, ulcers, nodules, or pustules
  2. Lesions start and/or are most pronounced on the head (muzzle, eyes), pinnae, and extremities (where sandflies feed) 
• Visceral leishmaniasis
  1. Mature dog in poor body condition with a rough haircoat 
  2. Chronic debilitation and often recurrent oculonasal discharge, with some crusting of the nose, and recurrent diarrhea
  3. Skin lesions occur in over 80% of dogs with visceral involvement
• Systemic signs: Fever, lethargy, cachexia, poor body condition/weight loss, rough haircoat, diarrhea, lymphadenomegaly, splenomegaly
  1. Can be highly varied and the following signs may appear alone or in combination: Recurrent oculonasal discharge, epistaxis, nasal crusting, claw abnormalities, glomerulonephritis, atrophic myositis of masticatory muscles, anterior uveitis, keratoconjunctivitis sicca, polyarthritis, chronic colitis, chronic hepatitis, vasculitis, myocarditis, osteomyelitis, orchiepididymitis, and/or meningoencephalomyelitis, locomotion disturbances
  2. Also a reported cause of failure of pregnancy
• A monoclonal or polyclonal gammopathy or signs renal insufficiency and nephrotic syndrome with azotemia and proteinuria/hypoalbuminemia may be present 

 

TYPICAL GROSS FINDINGS

• Cutaneous:
  1. Non-painful, non-pruritic focal or generalized dry exfoliative dermatitis with silvery white scales and alopecia 
     • And/or ulcerative dermatitis, multinodular dermatitis, mucocutaneous proliferative dermatitis, or papular dermatitis
     • Ulceration occurs especially over bony prominences and along mucocutaneous junctions
     • Periocular alopecia “lunettes” is common
  2. Hyperkeratosis and/or depigmentation, especially of nasal planum or foot pads
  3. Oncychogryphosis (hypertrophy and increased curvature of the claws) and/or paronychia (infection around the claw)
  4. Concurrent conjunctivitis, keratitis, uveitis, and/or blepharitis occurs in many dogs
  5. Lymphadenomegaly of multiple subcutaneous nodes is common
• Visceral:
  1. Lymphadenopathy, splenomegaly (“meaty spleen”), hepatomegaly
  2. Other gross signs: Tan mottling of lungs; liver granulomas; darkened kidneys; reddened bone marrow; non-erosive polyarthritis, polymyositis, osteolysis and osteoarthritis, proliferative periostitis, intestinal wall thickening, pancreatitis, meningitis, or chronic colitis
  3. Nodular and ulcerative oral lesions, including glossitis, in dogs (Blume et al, J Comp Pathol 2019)
  4. Case report: laryngeal granuloma in a French bulldog (Torrent et al, J Comp Pathol 2018)
  5. Repro:
     • Proliferative lesions of the penis and prepuce, protozoal orchitis, prostatitis 
     • Placental lesions with large numbers of amastigotes within trophoblasts of the zonary portion of the placenta

 

TYPICAL MICROSCOPIC FINDINGS 

• Macrophages predominate, but lymphocytes, plasma cells, and occasional multinucleated giant cells may be present
• Free or intracellular amastigotes: Oval to round, 2-4µm with a round, basophilic nucleus and a small, rod-like kinetoplast (oriented perpendicular to the nucleus); amastigote is often within a round, clear, intracellular parasitophorous vacuole
  1. Can also be within other leukocytes, endothelial cells, or fibroblasts
• Cutaneous:
  1. Orthokeratotic and parakeratotic hyperkeratosis are usually prominent
  2. The 3 most common patterns are granulomatous perifolliculitis, superficial and deep perivascular dermatitis, and interstitial dermatitis. 
     • Other patterns reported include lichenoid interface dermatitis, nodular dermatitis, lobular panniculitis, suppurative folliculitis, and intraepidermal pustular epidermitis
  3. Sebaceous adenitis with total obliteration of the sebaceous glands occurs in ~45% of cases
• Visceral:
  1. Widespread histiocytic, plasmacytic, or lymphocytic inflammation, generally with intrahistiocytic amastigotes, most severe in spleen, liver, lymph nodes but can be elsewhere
  2. Spleen: Hemic-lymphatic hypertrophy with macrophage proliferation and focal granulomas; follicle hyperplasia early, progressing to follicle atrophy; sinuses occupied by amastigote-laden macrophages and plasma cells
     • Similar lesions can occur in lymph nodes
  3. Kidney: Glomerular disease (mesangioproliferative or membranoproliferative glomerulonephritis or glomerulosclerosis) and nonsuppurative interstitial nephritis 
  4. Cardiovascular: Lymphoplasmacytic or granulomatous myocarditis; secondary vasculitis
  5. Muscle: Mononuclear myositis, myonecrosis, fibrosis
  6. Ocular inflammation/organisms: Conjunctiva, limbus, ciliary body, iris, cornea, sclera
  7. Joint: Uncommonly causes purulent, erosive or non-erosive, mono- or polyarthritis
  8. Central nervous system: Rare reports of meningoencephalitis, vasculitis, myelitis

 

ADDITIONAL DIAGNOSTIC TESTS

• Blood smear: Leishmania amastigotes may rarely be present in peripheral blood neutrophils and less frequently monocytes (Oikonomidis et al., J Vet Diagn Invest 2019)
• Bone marrow smear: Macrophages containing amastigotes, occasionally present in neutrophils, eosinophils, or white blood cell precursors (Oikonomidis et al., J Vet Diagn Invest 2019)
• Cell block stained with HE and immunocytochemistry improve sensitivity over smear cytology alone (Guerra et. Al, J Comp Path, 2019)
• Giemsa stain: Highlights amastigote blue
• Anti-Leishmania antibodies on serology (IFA, ELISA); IFA can also be used to quantify treatment success (Bruno et al., J Vet Diagn Invest. 2019)
• IHC for Leishmania spp. antigen (Casanova et al, J Comp Pathol 2019)
• PCR
• Skin test: Leishmanin skin test (LST), a delayed-type hypersensitivity (DTH) reaction can be used to identify dogs with a cell-mediated immune response (Ordeix, et. Al, J Comp Path 2018), similar to the intradermal tuberculin test (I-M01).
• Positive serum ANA and RF titers unlikely associated with arthritis secondary to leishmania infection (Tsouloufi et. Al, J Vet Diagn Invest 2022)

 

DIFFERENTIAL DIAGNOSIS

• Gross:
  1. Sarcoptic mange (I-P06), demodectic mange (I-P07), seborrhea, pemphigus foliaceus (I-M26), cutaneous/systemic lupus erythematosus (I-M28), bacterial infection, superficial necrolytic dermatitis (I-M16), Zn-responsive dermatitis (I-M18), sebaceous adenitis, neoplasia
• Microscopic:
  1. Trypanosoma cruzi (C-P06, tissue phase/amastigote form): Amastigotes within cardiomyocytes; kinetoplast oriented parallel to the nucleus
  2. Histoplasma capsulatum: 2-5 um, intracellular, narrow-based budding; predominantly histiocytic inflammation
  3. Toxoplasma gondii: 2-6 um tachyzoites; necrosis
  4. Neospora caninum (I-P17): 4-7 um tachyzoites
  5. Cryptococcus neoformans (I-F08): 2-20 um, mucicarmine-positive capsule
  6. Blastomyces dermatitidis (I-F06): 10-20 um, broad-based budding
  7. Sporothrix schenckii (I-F07): 4-10 um, oval to cigar shaped yeast
  8. Sebaceous adenitis
  9. Granulomatous, pyogranulomatous diseases

 

COMPARATIVE PATHOLOGY

• Feline:
  1. Rare. Generally cutaneous disease without visceral involvement, one to multiple variably smooth, ulcerated, or scaly papules and nodules on the pinnae and muzzle
• Equids: 
  1. Cutaneous leishmaniasis has been reported in horses, mules, and donkeys
  2. Single to multiple crusted or ulcerated papules and nodules on the pinnae, head, and neck, or less commonly, the legs, scrotum, and penis
  3. Most commonly caused by Leishmania siamensis
  4. Autochthonous (originating locally; no travel history) cutaneous leishmaniosis caused by Leishmania (Mundinia) martiniquensis reported in a horse from Florida (Menezes RC et al., J Comp Pathol. 2019)
• Bovine: 
  1. Rare reports, including a cow from Switzerland
  2. Most commonly caused by Leishmania siamensis
  3. Lesions described on the muzzle, pinnae, udder, and leg; Histo: Numerous eosinophils and foamy macrophages
• NHP: Rare, reported by a number of Leishmania species in a number of NHP species
• ZEW:
  1. Reported in a variety of species 
  2. Canids: Reported in a variety of captive and wild animals, including bush dogs, crab-eating foxes, golden jackals, hoary foxes, and maned wolves 
  3. Red kangaroos, hyrax, bats, and agouti; hyrax and bats possible species reservoirs

 

REFERENCES

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