Histoplasmosis and multicentric lymphoma in a Nubian goat

Abstract

Following treatment for pneumonia, a 1-y-old female Nubian goat was presented because of a persistent fever for 3 mo and peripheral lymphadenopathy for 1 mo. Cytology and histology of the superficial cervical and prefemoral lymph nodes demonstrated a moderate-to-marked “left-shifted” lymphoid population, suggestive of lymphoma, and extremely rare extracellular, 2–4 µm, oval, basophilic yeast, consistent with Histoplasma capsulatum. On immunohistochemistry, >95% of the lymphocytes demonstrated positive cytoplasmic and membranous immunoreactivity for CD3. Histoplasma spp. urine antigen and serum antibody testing were positive and negative, respectively. Panfungal PCR and sequencing of DNA extracted from scrolls of formalin-fixed, paraffin-embedded tissue yielded matches to H. capsulatum with 99–100% identity. Given the poor prognosis and persistent pyrexia, the animal was euthanized. Postmortem examination confirmed concurrent multicentric, intermediate-size, T-cell, lymphoblastic lymphoma and histoplasmosis; lesions consistent with intestinal coccidiosis and suspected pulmonary Rhodococcus equi were also noted. Although dimorphic fungi have been described previously in goats, lesions of Histoplasma spp. had not been documented in this species, to our knowledge. Given the low disease burden, it is suspected that the lymphoma was primary, leading to an immunocompromised state and development of secondary, opportunistic infections.

Lymphoma is considered a common neoplasm in the goat, comprising 14–18% of tumors in this species; the median age reported is 3 y, with a range of 1–9 y.^10,11 Frequently, caprine lymphoma is described as diffuse or multicentric, and can have a similar clinical presentation to caseous lymphadenitis, with peripheral lymphadenopathy, weight loss, and pyrexia.¹⁰ Hence, clinical distinction is imperative for treatment and prognosis. Dimorphic fungal infections have also been described in goats.^4-6,8,18 These infections manifest as primary cutaneous, respiratory, mammary, and/or neurologic disease attributable to Blastomyces, Coccidioides, or Cryptococcus spp.^4-6,8,18 To our knowledge, the lesions of Histoplasma spp. have not been documented in goats. Herein, we describe a case of concurrent multicentric lymphoma and histoplasmosis in a Nubian goat. Lymphoma was documented in multiple lymph nodes, and Histoplasma spp. yeast were visualized in peripheral lymph nodes and further confirmed with both PCR and urine antigen testing.

A 1-y-old, 28.6 kg, female Nubian goat was presented to the Texas A&M University, Veterinary Medical Teaching Hospital, Food Animal Medicine and Surgery Service (College Station, TX) for evaluation of persistent fever for 3 mo and generalized lymphadenopathy for 1 mo following diagnosis and treatment for pneumonia. At the onset of lymphadenopathy, lymph node aspirate cytology was performed by the primary veterinarian and was interpreted as neutrophilic lymphadenitis with presence of bacterial cocci. Culture (nalidixic acid–novobiocin–actidione–potassium tellurite and brain–heart infusion broth and plates) of the lymph node at that time was negative. The herd was routinely tested and historically negative for caseous lymphadenitis, caprine arthritis encephalitis, brucellosis, and tuberculosis. Another goat on the farm was diagnosed with multifocal pulmonary Rhodococcus equi abscesses earlier in the year.

Physical examination at presentation revealed pyrexia (39.5°C [103.2°F]), thin body condition score (2 of 5), and moderately to severely enlarged peripheral lymph nodes. Splenomegaly and abdominal lymphadenopathy were identified on abdominal ultrasound, as well as an ~1-cm pulmonary nodule on thoracic radiographs. Complete blood count revealed moderate anemia (hematocrit 0.18 L/L; reference interval [RI]: 0.22–0.38 L/L) and moderate leukocytosis (28.9 × 10⁹/L; RI: 4.0–13.0 × 10⁹/L) characterized by moderate lymphocytosis and moderate monocytosis. A McMaster fecal egg count revealed coccidia (11,800 oocysts/g). Fine-needle aspirate cytology, Tru-cut biopsy, and aerobic and fungal cultures of the superficial cervical and prefemoral lymph nodes were performed.

Cytologic evaluation revealed a moderately left-shifted lymphoid population with extremely rare extracellular, round-to-oval, 2–4 µm, yeasts with an eccentric, purple, crescent-shaped nucleus surrounded by a thin clear halo, consistent with Histoplasma capsulatum (Fig. 1A). Histologic evaluation of the biopsy revealed a similar predominance of immature lymphocytes, suggestive of lymphoma. Gomori methenamine silver (GMS) and Gram staining of the histologic sections failed to identify any microorganisms. Aerobic and fungal cultures of these lymph nodes were also negative. Histoplasma spp. antigen enzyme immunoassay (MiraVista Diagnostics, Indianapolis, IN) performed on urine was positive at 0.83 ng/mL (RI: none detected). Histoplasma antibody immunodiffusion assay (MiraVista Diagnostics) on serum was negative for both H and M bands (RI: none detected). Given a presumed poor prognosis and persistent pyrexia, the animal was euthanized.

Figure 1.

Lymph nodes from a 1-y-old female Nubian goat. A. Moderately left-shifted lymphoid population in a fine-needle aspirate, with an increased number and proportion of immature (intermediate and large) lymphocytes and fewer plasma cells. There are rare extracellular, 2–4 µm, oval, basophilic yeast cells with a 1-µm clear capsule (arrow). Modified Wright stain. Original magnification 1,000×. B. Scattered macrophages in the lymph nodes contain a few intracellular, 2–4 µm, oval, basophilic yeast cells with a 1-µm clear capsule (arrow). H&E. Original magnification 600×. Inset: yeast cells stain dark gray to black, suggestive of fungal origin. GMS stain. Original magnification 600×. C. Lymph node architecture is effaced by neoplastic intermediate-size lymphocytes. H&E. Original magnification 20×. Inset: the majority (>95%) of the lymphoid cells have cytoplasmic and membranous golden-brown staining, indicating T-cell origin. CD3 immunohistochemistry. Original magnification 400×.

Postmortem gross anatomic findings included diffusely enlarged, firm, tan lymph nodes that on cross-section were homogeneous tan with loss of internal structure; diffusely firm spleen with numerous 1–3-mm tan nodules; 2–3-mm tan nodules throughout the mucosa of the jejunum; and a 1.5-cm firm, tan nodule in the left caudal lung lobe.

Histologic evaluation of the prefemoral and scapular lymph nodes revealed a densely cellular, infiltrative, poorly demarcated, unencapsulated neoplasm composed of sheets of round cells effacing the lymph node architecture, obliterating the subcapsular sinuses, frequently extending through the capsule, and expanding the medullary sinuses as well as often within the lumen of intranodal blood vessels. Neoplastic cells had a small amount of eosinophilic cytoplasm, distinct cell borders, and round-to-irregular intermediate-size nuclei (3–4 times the diameter of a red blood cell) with finely stippled chromatin and 1–3 variably distinct nucleoli. Anisocytosis and anisokaryosis were moderate. There was occasional single-cell necrosis or apoptosis, bizarre mitotic figures, and a mitotic count of 150 (average of 15 mitotic figures per 40× field).

Also within the prefemoral and scapular lymph nodes, scattered macrophages contained phagocytized pyknotic debris and, rarely, small numbers of intracellular, oval, 2–4 µm, basophilic yeast cells with a 1-µm clear capsule (consistent with H. capsulatum) that were highlighted with GMS (Fig. 1B). The majority (>95%) of the lymphocytes were positive for CD3 (Dako/Agilent, Santa Clara, CA) and negative for CD20 (Thermo Fisher Scientific, Rockford, IL) on immunohistochemistry (Fig. 1C); primary antibody (CD3 or CD20) binding was detected through use of a secondary polymer detection reagent (Mach2 rabbit; Biocare Medical, Pacheco, CA) and a chromogen (diaminobenzidine) then counterstained with Mayer hematoxylin. Panfungal PCR (Texas A&M University Dermatopathology Specialty Service, College Station, TX) targeting the second ribosomal internal transcribed spacer (ITS2) region of the fungal genome was performed on scrolls of formalin-fixed, paraffin-embedded lymph node, as described previously.¹² Comparing 240 base pairs of the resulting PCR sequence contig to the National Center for Biotechnology Information database with the nucleotide Basic Local Alignment Search Tool yielded matches to H. capsulatum with 99–100% identity.

Histology of additional lymph nodes (hepatic, ileocolic, mandibular, popliteal, retropharyngeal, and tracheobronchial), bone marrow, liver, lungs, spleen, and small intestine displayed a similar neoplastic infiltrate, confirming disseminated lymphoma. Given the scarcity of yeast and financial limitations, GMS staining was only performed on the lymph nodes that demonstrated the organism on cytologic examination. The small intestinal mucosal nodules observed grossly were the result of coccidiosis, with mucosal hyperplasia and intralesional microgametes, macrogametes, and oocysts. The pulmonary nodule was a pyogranuloma with numerous intracellular gram-positive bacteria; preferred etiologic agents included R. equi and Corynebacterium pseudotuberculosis. R. equi was the most likely cause of the pulmonary pyogranuloma, given the history of previous infection in the herd (the herdmate was included in a 2018 study of R. equi infection in goats)¹⁹; however, culture and PCR could have been used for definitive characterization.

Caprine lymphomas are considered predominantly T cell and frequently appear in a multicentric distribution, often with mediastinal involvement.^10,11 A 2017 publication proposed a scheme to classify caprine lymphoma by applying a slightly modified version of the World Health Organization (WHO) classification system adapted for canine lymphoma.¹⁰ Based on this classification scheme, our case would be considered a multicentric, diffuse, intermediate-size, T-cell lymphoblastic lymphoma with a high mitotic count. To date, there are no definitive factors implicated in the development and manifestation of lymphoma in the goat. Retroviral infection (e.g., bovine leukemia virus, BLV) could be involved,¹⁵ along with possible environmental or genetic factors. We did not perform BLV testing in our case.

Dimorphic fungi described previously in goats include Blastomyces dermatitidis, Coccidioides immitis, Cryptococcus albidus, Cryptococcus gattii, and Cryptococcus neoformans; these infections manifested as cutaneous, respiratory, mammary, and/or neurologic disease.^4-6,8,18 Although seropositivity to H. capsulatum has been documented in a cohort of dairy goats in Brazil,⁷ the lesions caused by this dimorphic fungus have not been demonstrated microscopically or molecularly in this species to our knowledge. We identified the yeast on cytology and histology to be most consistent with H. capsulatum; however, Sporothrix and Emmonsia spp. (in particular the more recently described Emmonsia spp. that produce thermally dependent yeast-like organisms rather than classical large adiaspores)¹⁷ can display a similar morphology and have been shown to demonstrate cross-reactivity on the Histoplasma antigen assay (Martinez CR, Bohn AA. ASVCP mystery slide case 12. Proc 2016 ACVP & ASVCP Ann Meeting; 2016 Dec 3–7; New Orleans, LA).¹ Given the morphology noted on both cytologic and histologic evaluation, in conjunction with a positive antigen test and PCR results, we believe the yeast in our case to be H. capsulatum.

Although the development of lymphoma and infections (bacterial, parasitic, and fungal) could be coincidental conditions in our animal, we believe this to be evidence for an overall immunocompromised state. Immunocompromised patients can be susceptible to secondary or opportunistic infections as a result of impaired physical, humoral, and/or cellular defense mechanisms.^2,14 The concurrent findings of lymphoma and histoplasmosis, in particular, have been documented in humans and dogs.^3,9,13 There are 2 major hypotheses for the development of this disease pattern: 1) lymphoma developed first, leading to subsequent altered immune functionality and predisposition to infection, or 2) the fungal infection occurred first and chronic inflammation potentially led to an exuberant reactive and/or hyperplastic lymphoid response (which can mimic lymphoma) or malignant transformation of lymphoid cells.^3,9,13 Of note, fungal infections in immunocompromised patients are most often attributed to impaired cell-mediated immunity (mediated by T lymphocytes and macrophages).^2,14 Given the low microbial burden in this goat, it is probable that lymphoma was the primary issue and the infections occurred secondarily. The negative Histoplasma antibody immunodiffusion assay could be explained by antibody levels lower than the limit of detection by the assay, acute infection, ineffective antigen processing by neoplastic lymphocytes, and/or a diminished non-neoplastic lymphocyte population leading to ineffective immune response.^16,20 Antibody detection by enzyme immunoassay could have been beneficial in our case, as it has shown improved sensitivity (compared with immunodiffusion) and allows for semiquantitative detection of IgG and IgM antibodies.¹⁶ Although such testing is available commercially, it has not been validated in goats.

The concurrent finding of lymphoma and histoplasmosis supports an immunocompromised state; we speculate that the lymphoma was the primary event and that lymphoma facilitated secondary infections. Although secondary, infections can seriously complicate recovery from the primary disease. Our case demonstrates the importance of thorough evaluation for an underlying concomitant process in an animal with non-resolving disease.