Signalment:  

1-year-old castrated male domestic shorthair (Felis catus).The cat initially was presented to the referring veterinarian for anorexia and weight loss; upon examination, the cat had a fever, sneezing, and ocular and nasal discharge. The referring veterinarian prescribed prednisolone and doxycycline. Two weeks later, the cat was presented to the Kansas State University Veterinary Health Center. The cat was thin, and had fever, increased respiratory effort, peripheral lymphadenopathy, and ocular discharge. FIV-/FeLV and FCoV testing was negative. The cat was treated with blood transfusions, antibiotics, steroids, and oxygen supplementation, but worsened over the next two days. The cat was subsequently euthanized and sent to necropsy.

Gross Description:  

The cat was emaciated with diffuse pallor of the mucous membranes. The frontal sinuses were filled with thick yellow mucus. The submandibular, tracheobronchial, and mesenteric lymph nodes were diffusely enlarged, and the tracheobronchial lymph nodes were mottled dark-red and white. The lungs were diffusely orange-red and meaty with white miliary nodules. All lung sections sank in formalin.

Histopathologic Description:

Lymph node: The subcapsular and medullary sinuses of the lymph node are diffusely expanded by proteinaceous fluid and large numbers of macrophages. In the medullary sinuses, macrophages contain abundant hemosiderin, and there is frequent erythrophagocytosis. Occasional multinucleated giant cells are present, bearing up to 5 nuclei. Macrophages frequently contain intracytoplasmic 2-4 um diameter yeasts consisting of a 1-2 um diameter basophilic nucleus and a 1-2 um clear halo (capsule). The cortex of the lymph node contains numerous active germinal centers.

Large numbers of macrophages with intracytoplasmic yeasts are also present in the lungs (alveolar interstitium and lumina), spleen (red pulp), liver (portal tracts and sinusoids), kidney (perivascular interstitium), intestine (lamina propria and Peyer’s patches), and bone marrow (diffuse), as well as circulating macrophages in the brain and heart vasculature. A GMS stain revealed abundant intracytoplasmic 2-4 µm diameter yeasts.

Morphologic Diagnosis:  

Lymph node; Granulomatous lymphadenitis, diffuse, severe, with numerous intra-histiocytic yeasts consistent with Histoplas-ma capsulatum var capsulatum

Lab Results:  

CBC revealed minimally regenerative anemia, leukopenia consisting of neutropenia with toxic changes and increased bands, and thrombocytopenia. Serum chemistry revealed hyperglycemia, hypoalbuminemia, decreased anion gap, decreased ALT and ALP, and increased bilirubin.

 

A bone marrow aspirate showed high nucleated cellularity with rare precursor cells and abundant macrophages containing numerous round to oval yeast bodies (~2-4 microns in diameter) that have a thin outer halo with an eccentrically placed, crescent shaped, basophilic nucleus. The findings were consistent with histoplasmosis.

Condition:  

Granulomatous lymphadenitis/Histoplasma capsulatum

Contributor Comment:  

Histoplasma capsulatum is a dimorphic fungus that exists in a parasitic yeast form called “Histoplasma capsulatum”, and an environmental mycelial form called “Ajellomyces capsulata”.⁷ The organism is endemic in the St. Lawrence, Ohio, and Mississippi River valleys.⁶ It is soil-borne and prefers nitrogen-rich organic matter such as bird and bat excrement.³ The disease is non-contagious and affects humans as well as a wide variety of animals.⁷ Infection is usually subclinical without signs or lesions, and can result in a latent state.³; however, clinically evident disseminated infection can occur with immunosuppression and/or heavy infectious dose, and usually results in death.³ In this case, the cat was treated with steroids, which most likely predisposed it to the severe disseminated form of the disease.

Infection begins with ingestion or inhalation of soil contaminated with Histoplasma microconidia.⁴ Pulmonary or gut-associated macrophages phagocytose the microconidia, which germinate into the yeast form and replicate within phagosomes.⁷ In immuno-competent animals, Th1 cell-mediated immune responses eliminate the infection; in immunosuppressed states, the yeast-laden macrophages disseminate widely via the lymphatics and blood vessels to lymph nodes, liver, bone marrow, spleen, adrenal glands, etc.⁷

Histoplasma capsulatum evades the host’s extracellular immune defenses by residing intracellularly within macrophages, and by concealing immunostimulatory beta-glucan cell wall components with non-immuno-stimulatory alpha-glucans.^5,7 The yeast is internalized via macrophage complement receptors such as CR3 and CR4 while avoiding activation of pro-inflammatory receptors such as TLR2 and TLR4.⁵ Once inside the phagosome, the fungus produces antioxidant enzymes including superoxide dismutase Sod3 and catalases CatB and CatP.^5,7 It also prevents acidification of the phagosome and fusion of lysosomes by as yet unknown mechanisms.⁵ Other described virulence factors are involved with nutrient acquisition and production, including iron-scavenging hydroxamate siderophores, calcium-binding protein Cbp1, and various nucleic acid- and vitamin-synthesizing enzymes.^5,7

Clinical signs include fever, malaise, emaciation, diarrhea, nonregenerative anemia, and respiratory distress.^2-4 Gross lesions include enlargement or thickening of affected organs – including lymph-adenopathy, splenomegaly, hepatomegaly, thickened bowel walls, and interstitial pneumonia.^2-4 Histologically, organ in-volvement is characterized by granulo-matous infiltration with large numbers of mononuclear phagocytes bearing character-istic intracellular yeasts, as well as variable numbers of lymphocytes, plasma cells, and multinucleated giant cells.^3,4 Histochemical stains such as silver stains, periodic acid-Schiff, or Giemsa stains can highlight yeast organisms. For antemortem diagnosis, cytology from rectal scrapings or fine needle aspirates from lymph nodes and internal organs can also demonstrate intrahistiocytic yeasts.^3,4,6 Fungal culture is hazardous, however, due to infectivity of microconidia produced in the mycelial phase. Ketoconazole, itraconazole, and fluconazole alone or in combination with amphotericin B has been used to successfully treat dogs and cats.^2-4 Itraconazole is the recommended treatment of choice.^3,4

JPC Diagnosis:  

1. Lymph node:  Lymphadenitis, granulomatous, multifocal to coalescing, marked, with mild reactive lymphoid hyperplasia and sinus edema.

2. Lymph node: Draining hemorrhage with hemosiderosis.

Conference Comment:  

The contributor provides an excellent summary of the virulence factors produced by Histoplasma capsulatum to evade the host immune response and produce severe disease. Post-exposure, the infected host requires both innate and adaptive immunity to neutralize the pathogen and overcome the virulence factors mentioned above.⁵ Macrophages rapidly phagocytose inhaled conidia and subsequently stimulate pro-inflammatory cytokines such as IFN- γ, TNF-α, and IL-12 associated with a Th1 cell mediated response.^5,7 Macrophages also produce superoxide radicals, nitric oxide and other lysosomal enzymes to destroy the phagocytosed pathogen.¹ However, as men-tioned by the contributor, Histoplasma capsulatum evades destruction within the phagolysosome and can survive, replicate, and disseminate to multiple tissues within macrophages.⁵ In this case, conference participants noted several instances of characteristic narrow-based budding of the organism within macrophages. 

Conference participants also discussed dendritic cells as an important component of the innate immune response to this or-ganism. Dendritic cells typically phagocytose and destroy the organism with higher efficacy than macrophages. In addition, they present antigens to naïve CD4+ and CD8+ lymphocytes via MHC-1 to generate a T cell mediated immune response and destroy infected cells.¹ Cell-mediated immunity (CMI) is crucial for the host to clear the organism and T cells, as the central effectors CMI, are necessary to degrade the pathogen.^1,5,7 Severe disease occurs in immunosuppressed animals as well as hosts with a bias toward a Th2 humoral response. Humoral immune responses generally have a limited role in the clearance of intracellular pathogens.^5,7

The nature of the inflammation within the lymph node generated spirited discussion during the conference, as it has so many times before over the many years of the Wednesday Slide Conference. Some con-ference participants preferred histiocytic, rather than granulomatous lymphadenitis. Those favoring the term “histiocytic” noted the paucity of multinucleated giant cells and lack of activated fibroblasts surrounding aggregates of infected macrophages, histologic features often associated with the more typical granulomatous response. Due to variation in sections, others noted more numerous multinucleated giant cell macro-phages surrounding accumulated epithelioid macrophages and preferred granulomatous lymphadenitis. Conference participants also posited that the subcapsular location of many of the aggregating macrophages may be secondary to antigen presentation within pre-existent lymphoid follicles. This would stimulate follicular lymphoid hyperplasia and medullary plasmacytosis with Russell body-laden Mott cells, as seen in this case.

References:

1. Ackerman M. Inflammation and healing. In: McGavin MD, Zachary JF, eds. Pathologic Basis of Veterinary Disease. 5th ed. St. Louis, MO:Mosby Elsevier; 2012: 89-146.

2. Aulaka HK, Aulakh 2. KS, Troy GC. Feline histoplasmosis: a retro-spective study of 22 cases (1986-2009). J Am Anim Hosp Assoc. 2012; 48(3):182-187. 

3. Brömel C, Greene CE: Histo-plasmosis. In: Greene CE, ed. Infectious Diseases of the Dog and Cat. 4th ed. St Louis, MO: Elsevier Saunders; 2012:614-621. 

4. Brömel C, Sykes JE. Histoplasmosis in dogs and cats. Clin Tech Small Anim Pract. 2005; 20(4):227-32.

5. Garfoot AL, Rappleye CA. Histoplasma capsulatum surmounts obstacles to intracellular path-ogenesis. FEBS Journal 2015; 283:619-633

6. Kerl ME: Update on canine and feline fungal diseases. Vet Clin North Am Small Anim Pract 2003; 33:721-747.

7. Woods, JP. Revisiting old friends: Developments in understanding Histoplasma capsulatum pat-hogenesis. J Microbiol 2016; 54:265-276.

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2-1. Lymph node, cat.

2-2. Lymph node, cat.

2-3. Lymph node, cat.

2-4. Lymph node, cat.

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