JPC SYSTEMIC PATHOLOGY

URINARY SYSTEM

January 2024

U-P03

 

Signalment (JPC Accession # 1752289): Mixed breed dog

 

HISTORY: Tissue from a mixed breed dog with a two-year history of chronic dermatitis with pruritus. Before death, the animal had periodic epistaxis.

 

HISTOPATHOLOGIC DESCRIPTION: (U-P03a) Kidney: Multifocally expanding approximately 10% of the renal interstitium, separating and surrounding cortical tubules and glomeruli, and expanding the pelvic subepithelial connective tissue, are numerous plasma cells and macrophages, fewer lymphocytes and neutrophils, and occasional lakes of homogenous eosinophilic material (proteinaceous fluid). Macrophages are often expanded by numerous intracytoplasmic 3-4 µm round to oval protozoa, which contain a 1-2 µm diameter, basophilic nucleus with an adjacent perpendicular basophilic kinetoplast (amastigotes). Multifocally, glomeruli often have: cuboidal parietal epithelium lining Bowman’s capsule (hypertrophy); surrounded by marked amounts of fibrous connective tissue (periglomerular fibrosis); glomerular tufts adhered to the parietal epithelium (synechia); increased numbers of mesangial cells and glomerular capillary loops thickened by eosinophilic, homogenous material (membranoproliferative glomerulonephritis); amastigotes within mesangial cells or macrophages; and uriniferous space expanded by variable amounts of eosinophilic homogenous material (proteinosis). Multifocally, proximal tubules and collecting ducts have one or more of the following changes: lined by attenuated epithelium; mildly ectatic and contain moderate amounts of proteinaceous material (tubular proteinosis); and rarely contain a basophilic, granular material (mineralization). Multifocally, the subepithelial pelvic connective tissue is expanded by the previously described inflammatory cells including infected macrophages and the overlying epithelium is mildly hyperplastic with occasional transmigration by neutrophils.  

 

U-P03b: Kidney (Giemsa): There is multifocal positive purple staining of amastigote nuclei and adjacent kinetoplasts.

 

MORPHOLOGIC DIAGNOSIS: Kidney: Nephritis, interstitial, lymphoplasmacytic and histiocytic, multifocal, moderate, with glomerulonephritis, and numerous intrahistiocytic amastigotes, mixed breed, canine.

 

ETIOLOGIC DIAGNOSIS: Renal leishmaniasis

 

CAUSE: Leishmania sp. 

 

CONDITION: Visceral leishmaniasis

 

SYNONYMNS: Kala-azar, Dum dum fever

 

 

GENERAL DISCUSSION: 

• Zoonotic disease with cutaneous and systemic manifestations caused by obligate intracellular diphasic protozoa
• Chronic disease caused by obligate intrahistiocytic, diphasic (promastigotes, amastigotes) protozoan parasite
• Affects skin, lymph nodes, spleen, kidney, liver, bone marrow, heart, gastrointestinal tract, and lungs
• Dogs are natural hosts and the primary domestic reservoir for human infection
• Transmission by:
  • Bite of female sandfly (Phlebotomus spp. [Old World], Lutzomyia spp. [New World]) or stable fly (Stomoxys spp.)

• Rarely transmitted by blood transfusions, venereal, and transplacental

• There are three forms of the disease:
  1. Cutaneous leishmaniasis (I-P15) (usually L. infantum, L. donovani, L. braziliensis spp. complex, or L. mexicana complex)
     • Sandfly bites around muzzle, ears, eyes
     • Exfoliative to ulcerative, granulomatous
  2. Mucocutaneous leishmaniasis (usually L. braziliensis)
     • Central America 
     • Animals not frequently infected in nature
     • Canine nodular mucosal leishmaniasis of the mouth, tongue, nose and penis
  3. Visceral leishmaniasis (U-P03, H-P07) (usually L. infantum, L. donovani)
     • Wide geographic distribution
     • Mimics histoplasmosis
     • Endemic in the US: outbreaks of L. infantum (chagasi) primarily in foxhounds in U.S. and Canada
     • Skin lesions occur in >80% of dogs with visceral involvement

PATHOGENESIS:

• Severity of disease determined by host immune response 

• Th-1 response > resistant (TNF-α, IL-2 and IFN-y mediate protective response)
• Th-2 response > susceptible; chronicity may lead to antigen/antibody complexes
• Chronic disease leads to T-lymphocyte depletion and B cell proliferation; infected macrophages are also lysed by CD8+ cytotoxic T cells; 

• Excess B cells generate Ig
  • Immune-mediated thrombocytopenia (IgG driven)
  • Circulating immune complexes (CIC) > vasculitis, polyarthritis, uveitis, glomerulonephritis; Chronic proteinuric nephritis due to CIC deposition in kidneys is the leading cause of death in dogs with leishmaniasis  

 

LIFE CYCLE:  

• Sandfly takes a blood meal from infected mammalian host > ingests mononuclear cells containing amastigotes (Leishman‑Donovan bodies) > amastigotes multiply by binary fission in the mid-gut > transform into flagellated promastigotes > sandfly feeds and injects many promastigotes into host (transmission) > phagocytized by macrophages > revert to non-flagellated amastigotes (leishmanial form) > begin multiplying by binary fission locally (cutaneous/ mucocutaneous forms) or throughout reticuloendothelial system (visceral form) > rupture out of macrophage > infect new cells
• Prolonged incubation period of 3 months – 7 years after sandfly bite
• Reservoirs: Dogs, cats, carnivores, rats, rock hyraxes, potentially bats

 

TYPICAL CLINICAL FINDINGS:

• Asymptomatic proteinuria, which often is the only indication of renal involvement, may predispose to arterial thromboembolism, muscle wasting and cachexia
• Nonspecific: Fever, lethargy, cachexia, poor body condition, rough haircoat
• Nonpruritic, exfoliative generalized dermatitis, most severe in facial, aural, and periocular regions, and onychogryphosis
• Secondary infections are common (demodicosis, pyoderma, pneumonia) 
• Coagulopathy – epistaxis due to secondary immune-mediated thrombocytopenia
• Systemic hypertension

 

CLINICAL PATHOLOGY:

• Proteinuria, azotemia, elevated creatinine
• Anemia, non-regenerative (bone marrow infection; secondary myelodysplastic syndrome)
• Thrombocytopenia (immune mediated by IgG)
• Elevated total protein, hypergammaglobulinemia (IgG, polyclonal or monoclonal), hypoalbuminemia
• Nephrotic syndrome (proteinuria, hypoalbuminemia, hyperlipidosis, peripheral edema)

 

TYPICAL GROSS FINDINGS:  

Cutaneous:

• Alopecia, ulcers, nodules, or pustules
• Exfoliative dermatitis with silvery-white scales 
• Most severe on muzzle, periorbital (“periocular lunettes”), and aural regions (where sandflies feed); nodular mucosal leishmaniasis is also reported
• Lymphadenopathy
• Onychogryphosis (hypertrophy and increased curvature of the claws) with mild to severe interface dermatitis

Visceral:

• Generalized lymphadenopathy, hepatosplenomegaly
• Liver and spleen are enlarged and dark brown; liver contains numerous granulomas
• Kidneys usually normal contour but darker than normal
• Nodular and ulcerative oral lesions in dogs (Blume et al, J Comp Pathol 2019)

 

TYPICAL LIGHT MICROSCOPIC FINDINGS:

• Kidney: Glomerulonephritis from immune complex deposition in vessel walls 

• Glomerulosclerosis (without IC deposition), membranoproliferative or mesangioproliferative glomerulonephritis
• +/- Amyloidosis, and intrahistiocytic amastigotes 

• Liver: Numerous granulomas
• Splenic follicles are hyperplastic (early) or atrophied (advanced)
• Widespread plasmacytic, lymphocytic, or histiocytic inflammation, most severe in spleen, liver, lymph nodes, bone marrow (bone marrow up to 50% plasma cells)
• Atrophic myositis of masticatory muscles
• A recent study found that cardiac lesions, such as lymphoplasmacytic or granulomatous myocarditis (especially in the right atrium), myocardial necrosis and increased interstitial collagen, are prevalent in dogs with leishmaniasis (even if there are no clinical signs of cardiac disease)

• Intrahistiocytic amastigotes 

• Oval to round, 2.5 – 5 µm long, 1.5 to 2.0 µm wide
• Kinetoplasts (giant mitochondria) perpendicular to nucleus (red to purple on Giemsa) 
• Can occasionally be seen in other leukocytes, endothelial cells or fibroblasts

• Skin: Granulomatous perifolliculitis and perivascular dermatitis 

• Exfoliative dermatitis- orthokeratotic hyperkeratosis
• Papular dermatitis- nodular to diffuse pyogranulomatous dermatitis
• Onychogryphosis- lichenoid mononuclear dermatitis

• Less common: Myocarditis, polyarthritis, colitis, orchiepididymitis, ocular/periocular granulomatous inflammation, prostatitis, abortion/failure to become pregnant

 

ULTRASTRUCTURAL FINDINGS:  

• Often within parasitophorous vacuole, oval amastigote with a double membrane-bound nucleus 
• Kinetoplast is perpendicular to nucleus
• Flagellum in a flagellar pocket near nucleus

 

ADDITIONAL DIAGNOSTIC TESTS: 

• Lymph node, spleen, liver, or bone marrow aspirates; biopsy; IHC; ISH
  • Amastigote load seems to increase in the preferential feeding sites of sandflies; biopsies of the skin of the muzzle may have highest parasite yield
  • Renal cytology is characterized by mixed inflammation, clusters of renal tubular cells, and the presence of intrahistiocytic organisms.
• Cell-block technique
  • Aspirate is centrifuged, cellular pellet is fixed, paraffin embedded, and treated like a tissue specimen. Histology and IHC are then performed. (Guerra et al., J Comp Pathol 2019)
• Giemsa: Stains cytoplasm blue, nucleus red, and kinetoplast purple
• Anti-Leishmania antibodies on serology (IFA, ELISA)
  • May cross-react with sporotrichosis
• IHC for Leishmania spp. antigen (Casanova et al, J Comp Pathol 2019)
• PCR

 

DIFFERENTIAL DIAGNOSIS:

• Organisms that may appear similar microscopically:

• Trypanosoma cruzi (C-P06, tissue phase/amastigote form) – Intracellular, within pseudocysts primarily in muscle, especially heart muscle; multiply by binary fission; round to oval, 1.5-4µm, contain a nucleus, and a rod-like kinetoplast parallel to nucleus, no free flagellum or undulating membrane 
• Histoplasma capsulatum – intracellular; up to 10 cytoplasmic 2 X 3 µm in diameter yeast characterized by a clear, 1 µm wide halo surrounding a 1 µm basophilic center; replication is by narrow-based budding
• Histoplasma capsulatum var. farciminosum – see above
• Toxoplasma gondii – intracellular; numerous 2-4 µm, round, basophilic, surrounded by a thin clear ring (tachyzoites within a pseudocyst)
• Neospora caninum (I-P17) – intracellular; 2 x 5 µm lunate zoites arranged in small clusters (tachyzoites) and within cysts (bradyzoites); cysts are up to 40 um and have a thick eosinophilic hyalinized wall
• Neorickettsia helminthoeca – intracellular; gram negative; organisms are 0.3-2.0 µm, pleomorphic cocci or rods 
• Sporothrix schenckii (I-F07): 4-10um, oval to cigar shaped yeast

• Causes of monoclonal gammopathy: Multiple myeloma, lymphoma, leukemia, canine ehrlichiosis
• Causes of splenomegaly: Hemolytic anemia, salmonellosis, Anthrax sp., Mycoplasma spp., barbiturates, volvulus
• Causes of granulomatous bone marrow inflammation: Mycobacterium spp., systemic mycoses

 

COMPARATIVE PATHOLOGY:

• NHPs: Natural disease is caused by Leishmania major and L. braziliensis; cutaneous disease induced in many NHP species as an animal model of human disease
• Wild rodents: Reservoir host for cutaneous / mucocutaneous forms in humans
• Wild and domestic canids (foxes and jackals): Main reservoirs for visceral form in humans
• Cats, horses, mules, donkeys, and opossums: Susceptible but considered accidental hosts – rare, lesions similar to those in dog; crusted, ulcerated nodules on the pinna, head, and neck; granulomatous, lymphoplasmacytic dermatitis
• Wildlife: Also reported in wolves, genets, captive red kangaroos, hyrax, and bats
• Case report of cutaneous Leishmaniasis caused by Leishmania martiniquensis in a horse in florida (Menezes, J Comp Pathol 2019)

 

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