History

A 7.5-year-old 30.4-kg (66.9-lb) spayed female Collie was referred to the Veterinary Teaching Hospital at Michigan State University because of intermittent left forelimb lameness of 1.5 years' duration, which had progressed to continuous lameness during the preceding 5 months. Clinical signs were not ameliorated with anti-inflammatory drug administration (meloxicam, 0.1 mg/kg [0.045 mg/lb], PO, q 24 h).

Clinical, Gross, and Initial Cytologic Findings

At initial evaluation, the dog would not bear weight on the left forelimb, but occasional toe touching was evident; signs of pain increased with limb extension. Bilateral popliteal lymphadenomegaly was noted. Serum biochemical analyses revealed mildly high alanine aminotransferase (204 U/L; reference range, 14 to 102 U/L) and aspartate aminotransferase (37 U/L; reference range, 19 to 34 U/L) activities. Results of a CBC and urinalysis were unremarkable. Radiography of the left forelimb revealed osteolytic and periosteal proliferative lesions associated with the articular surface of the proximal portion of the left humerus. Thoracic radiography and abdominal ultrasonography revealed sternal, thoracic, iliac, and abdominal lymphadenomegaly.

A bone marrow aspirate sample was collected from the osteolytic and periosteal proliferative lesion of the proximal portion of the left humerus; slide preparations of the sample were stained and evaluated microscopically. The modified-Wright-stained bone marrow aspirate was moderately cellular and hemodiluted with low numbers of megakaryocytes as well as myeloid and erythroid progenitor cells. Moderate numbers of ruptured cells and rare intact cells with pale blue cytoplasm that had an amorphous to linear pattern were present (Figure 1).

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Photomicrograph of a fine-needle aspirate sample of bone marrow obtained from the left humerus of a Collie that was evaluated because of intermittent left forelimb lameness of 1.5 years' duration, which had progressed to continuous lameness during the preceding 5 months. The large intact cell has an intracytoplasmic amorphous to linear pattern. Modified Wright stain; bar = 10 μm.

Citation: Journal of the American Veterinary Medical Association 238, 1; 10.2460/javma.238.1.51

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Formulate differential diagnoses from the history, clinical and cytologic findings, and Figure 1—then turn the page →

Histopathologic and Additional Cytologic Findings

Fine-needle biopsy samples were obtained from the medial iliac and abdominal lymph nodes. Prepared slides were stained with modified Wright stain and examined microscopically. Findings (ie, low cellularity and moderate hemodilution) were similar to those for the bone marrow aspirate samples. Clumps of deteriorated cells with extracellular, nonpigmented, septate fungal hyphae (3 to 5 μm in diameter with parallel sides) with occasional dichotomous branching (at 90° angles; range, 60° to 100° angles) were evident (Figure 2). There also were moderate numbers of ruptured cells with an amorphous to linear pattern (similar to the suspected hyphae in the bone marrow aspirates) within residual cytoplasm. Moderate numbers of nondegenerate neutrophils, macrophages, and rare large multinucleated cells also were present.

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Photomicrograph of a fine-needle aspirate sample from an abdominal lymph node in the dog described in Figure 1. Notice the extracellular, septate fungal hyphae. Modified Wright stain; bar = 10 μm.

Citation: Journal of the American Veterinary Medical Association 238, 1; 10.2460/javma.238.1.51

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Bone marrow core sections from the osteolytic and periosteal proliferative lesion of the proximal portion of the left humerus were also collected, paraffin embedded, stained with H&E stain, and examined microscopically The sections were hypocellular overall. However, several focal areas consisting of numerous, large, epithelioid macrophages were detected; the macrophages had an intracytoplasmic pattern similar to that detected in cells in the bone marrow aspirate sample (Figure 3). Examination of bone marrow core sections that were stained with Gomori methenamine silver or periodic acid–Schiff stain revealed fungal hyphae within macrophages. There also were rare extracellular, nonpigmented, septate fungal hyphae (3 to 4 μm in diameter; 10 to 14 μm in length) with occasional dichotomous branching.

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Photomicrograph of a bone marrow core section obtained from the lesion of the left humerus in the dog described in Figure 1. Notice that the macrophage contains phagocytized amorphous to linear material. H&E stain; bar = 10 μm.

Citation: Journal of the American Veterinary Medical Association 238, 1; 10.2460/javma.238.1.51

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Microbial Findings

Samples from both the proximal portion of the left humerus and medial iliac lymph node were placed on blood and Sabouraud agar plates (2% Sabouraud dextrose agar); plates were incubated at room temperature and 37°C for 5 days. Colonies characterized microscopically by biverticillate conidiophores, which is typical of some members of the genus Penicillium, were obtained in pure culture (Figure 4). The colonies had an atypical yellow-brownish color (most Penicillium spp colonies are green). On the basis of classic taxonomic characteristics such as color, size, and shape of conidiophores, phialides, and conidia,¹ the organism was tentatively identified as Penicillium verruculosum.

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Photomicrographs of fungal colonies cultured from core bone marrow samples of the proximal portion of the left humerus in the dog described in Figure 1. In the inset, biverticillate conidiophores that are typical of Penicillium verruculosum are visible. Lactophenol blue stain; bar (applies also to inset) = 20 μm.

Citation: Journal of the American Veterinary Medical Association 238, 1; 10.2460/javma.238.1.51

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Morphologic Diagnosis

Systemic penicilliosis with neutrophilic and macrophagic lymphadenitis and granulomatous osteomyelitis.

Comments

Canine blastomycosis, histoplasmosis, coccidiomycosis, and aspergillosis are reported to occasionally cause systemic fungal infections that may include bone involvement.² There are also some single case reports^3–10 of infection with rare fungal species that are generally considered opportunistic. In the dog of this report, the preservation of the fungal hyphal morphology was poor (most hyphae were partially digested within macrophages) in routinely stained cytologic samples. On the basis of the fungal characteristics that were evident, likely differential diagnoses for branching, nonpigmented, septate hyphae included infection with Aspergillus spp, Penicillium spp, Fusarium spp, and Paecilomyces spp. The extracellular hyphae detected cytologically could easily be confused with Aspergillus spp; however, infrequent hyphal branching that lacked the characteristic 45° angle associated with that genus was more suggestive of Penicillium spp.

Speciation of fungal isolates is usually achieved via examination of fruiting structures formed in culture. In contrast, only hyphae are present in most cytologic and histologic samples, with the rare exception of fruiting heads within an air-exposed cavity.^11,12 Although precise fungal identification was not possible on the basis of the histologic and cytologic features of the hyphae in the dog of this report, cultures of lymph node and bone marrow core samples provided evidence of morphological features (eg, biverticillate conidiophores) that enabled classification of the fungus as P verruculosum. To confirm the diagnosis, PCR analysis of cultured colonies and bone marrow core samples was performed. The partial 18S small subunit rDNA sequence (490 base pairs) was amplified via a PCR procedure with NS1 and NS2 universal primers (according to standard protocols) and sequenced by use of dye-labeled teminators.^a By use of BLAST,^b it was determined that the amplicons had 100% agreement with published sequences from the 18S small subunit rDNA gene of some Penicillium spp, including P verruculosum (GenBank accession No. AF510496).

In cooler climates worldwide, 200 or more known species of Penicillium are found in soil, decaying vegetation, seeds, and grain¹³; most species are considered nonpathogenic (opportunistic) for humans and other animals.^13,14 In humans, invasive fungal infections have become increasingly common over the last 2 decades because of an increased prevalence of immunosuppression (attributable to AIDS, transplantation, chemotherapy, or corticosteroid use) and opportunity for foreign travel as well as recognition of infections in otherwise healthy individuals.¹⁵

In dogs, infections of the nasal cavity,^16,17 lungs,^4,5 lymph nodes,^5,6 and bones with Penicillium spp have been reported.^5–7 Species of penicillia that cause infection in dogs include P verruculosum, Penicillium purogenum, Penicillium commune, and Penicillium brevicompactum.^3,4,6,7 Unfortunately, some of the Penicillium isolates in those reports were identified only to the genus level, and most were speciated on the basis of culture morphology only without results of PCR amplification and nucleotide sequence analysis. Increasingly, evaluation of fungal culture morphology combined with PCR amplification and sequencing findings are used as the gold standard for fungal speciation; samples from a variety of sources can be used. However, results must be interpreted with caution because PCR procedures are highly sensitive (capable of generating false-positive results) and members of the genus Penicillium are common laboratory contaminants. However, for the dog of this report, Penicillium organisms were consistently grown in cultures of lymph node and bone marrow core samples and the morphological features of cultured isolates were consistent with those observed via cytologic and histologic examination of similar samples. In addition, results obtained via PCR analysis of paraffin-embedded bone marrow core sections and cultured colonies were identical.

The immune status of the dog of this report was unknown; in general, this information is not reported for most dogs with penicilliosis. Disseminated fungal infections appear to be related to local climate and to predisposing immune defects (associated with impaired cell-mediated immunity or immunosuppressive therapy) in the affected individual.^2,5,18 Interestingly, osteomyelitis caused by P verruculosum (diagnosed on the basis of morphology of the cultured organism alone) with multiple osteolytic or proliferative lesions distal to the carpus and tarsus in a dog has been previously reported.⁷

The dog of this report received antifungal treatment following diagnosis, but left forelimb lameness persisted; subsequently, lethargy, anorexia, and hind limb weakness developed. Euthanasia was elected because of the dog's deteriorating condition.