Pathology in Practice

Paola Cazzini Department of Pathology, College of Veterinary Medicine, University of Georgia, Athens, GA 30602.

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Melinda S. Camus Department of Pathology, College of Veterinary Medicine, University of Georgia, Athens, GA 30602.

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Bridget C. Garner Department of Pathology, College of Veterinary Medicine, University of Georgia, Athens, GA 30602.

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History and Clinical Findings

A 4-year-old 18.6-kg (40.9-lb) sexually intact male mixed-breed dog was evaluated at the University of Georgia because of acute blindness. For 3 months prior to the evaluation, the dog had had intermittent left forelimb lameness. Treatment with various antimicrobials and administration of prednisone over a 2-week period (as prescribed by the referring veterinarian) did not lead to resolution of the lameness. Immediately prior to initial evaluation at the University of Georgia, the dog suddenly became blind. On ophthalmic examination, the dog was blind in the left eye but had vision in the right eye. Although the dog had vision on the right side, bilateral optic lesions including severe panophthalmitis with marked anterior uveitis, retinal detachments, and intraretinal hemorrhages, were detected. A CBC revealed no abnormalities. Moderate hyperglobulinemia (4.4 g/dL; reference interval, 2.5 to 2.9 g/dL) was identified, but the remaining biochemical findings were unremarkable. A urine sample collected via cystocentesis was also analyzed and revealed a specific gravity of 1.016, traces of protein and bilirubin, and 10 to 50 WBCs/hpf (40×). Rare bacteria and yeast organisms were seen during examination of a wet-mount preparation of the urine sediment. A firm swelling of the left elbow region was also noted, and radiography of the affected area revealed a palisading periosteal proliferation surrounding the distal diaphysis of the humerus with overlying soft tissue swelling. Fine-needle aspirate specimens of the bone lesion were collected for cytologic examination (Figure 1). The dog was euthanized shortly thereafter because of its deteriorating clinical status and poor prognosis. A necropsy was not performed.

Figure 1—
Figure 1—

Photomicrograph of a fine-needle aspirate specimen of a proliferative bone lesion at the distal diaphysis of the left humerus of a 4-year-old mixed-breed dog that had had intermittent left forelimb lameness of 3 months’ duration and had suddenly become blind prior to evaluation. Modified Wright stain; bar = 50 μm.

Citation: Journal of the American Veterinary Medical Association 242, 8; 10.2460/javma.242.8.1079

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

Cytologic and Histologic Findings

Cytologic examination of modified Wright-stained fine-needle aspirate specimens obtained from the bone lesion revealed highly cellular specimens containing mildly degenerate neutrophils admixed with numerous foamy and epithelioid macrophages. Moderate numbers of spindle-shaped cells, interpreted to be reactive fibroblasts, were also seen. Extracellular, round yeasts were admixed with the inflammatory cells, and numerous intracellular yeasts were present within macrophages. The yeasts, which ranged from 2 to 20 μm in diameter, had a thick, nonstaining capsule surrounding a small, purple nucleus. Occasional narrow-based budding of the organisms was observed.

Histologic examination of a wedge of bone obtained from the affected site after euthanasia of the dog revealed diffuse pyogranulomatous inflammation composed of epithelioid macrophages and neutrophils, with fewer lymphocytes and plasma cells, which expanded the periosteum and effaced the medullary cavity. The inflammatory response was centered on extracellular and intracellular organisms similar to those detected in the fine-needle aspirate specimens. Pale, eosinophilic, necrotic bone, with empty lacunae and remodeling characterized by reversal lines, was also prominent (Figures 2 and 3).

Figure 2—
Figure 2—

Photomicrograph of a section of a bone wedge obtained from the affected region of the left humerus of the dog in Figure 1. Notice the severe pyogranulomatous inflammation admixed with cryptococcal yeasts. Bone necrosis and remodeling were also present. H&E stain; bar = 100 μm.

Citation: Journal of the American Veterinary Medical Association 242, 8; 10.2460/javma.242.8.1079

Figure 3—
Figure 3—

Photomicrograph of a section of a bone wedge obtained from the affected region of the left humerus of the dog in Figure 1. Cryptococcal yeasts have been phagocytized by macrophages and are also observed in extracellular locations. Many foamy macrophages and fewer neutrophils are present. H&E stain; bar = 50 μm Inset—Cryptococcal organisms with narrow-based budding. H&E stain; bar = 10 μm.

Citation: Journal of the American Veterinary Medical Association 242, 8; 10.2460/javma.242.8.1079

Additional Laboratory Findings

Aerobic bacterial culture of a sample of the collected urine yielded no growth. Analysis of a serum sample collected at the initial evaluation revealed that the cryptococcal antigen titer was 400. A titer ≥ 8 is considered to indicate a positive result of the assay for Cryptococcus neoformans antigen. The serum sample was negative for antibodies against Babesia canis via indirect immunofluorescence assay and for antibodies against Blastomyces dermatitidis and Histoplasma capsulatum via agar gel immunodiffusion testing.

Morphologic Diagnosis and Case Summary

Morphologic diagnosis: marked pyogranulomatous osteomyelitis with intra- and extracellular yeasts morphologically compatible with Cryptococcus sp.

Case summary: cryptococcal osteomyelitis in a dog.

Comments

Cryptococcosis is an important disease in humans and animals. It affects predominantly cats, in which it is the most common systemic mycosis.1 In dogs, the frequency with which cryptococcosis develops is one seventh to one tenth the frequency that it develops in cats.1,2 Cryptococcal organisms are yeasts that have a polysaccharide capsule (1 to 25 μm in thickness) and a round or oval nucleus (4 to 15 μm in diameter) and that divide by narrow-based budding.3 The genus Cryptococcus can be divided into 2 groups: the Cryptococcus neoformans and Cryptococcus gattii group and the non–C neoformans group. Cryptococcus neoformans and C gattii are the most pathogenic. Even though the Cryptococcus spp in the non–C neoformans group are generally considered saprophytes, Cryptococcus laurentii, Cryptococcus magnus, and Cryptococcus albidus have been recently identified as pathogens in domestic animals.4

Cryptococcal infection is thought to be acquired from the environment, and no cases of disease transmission from an affected animal to another have been reported.1 Cryptococcus neoformans is distributed worldwide and can be found in birds’ feces (most commonly pigeons) and in decaying plant matter. This fungal species typically affects immunocompromised hosts.

The pathogenesis of disease caused by C neoformans is not completely understood. However, it is hypothesized that the spores are inhaled1,5 and reach the alveolar spaces, where they are phagocytized and neutralized by activated macrophages.5 If a defect or delay in macrophage activation occurs, then the spores germinate into encapsulated yeasts. The capsule inhibits phagocytosis and resists phagosome digestion, thereby making the microorganism more resistant to the host's immune defenses.5 Capsular material also acts directly against the host's cells because polysaccharides from the capsule promote dysfunction and lysis of macrophages, and the capsular material can induce apoptosis.6 Subsequent dysfunctions in T lymphocytes and alveolar macrophages allow the organisms to proliferate in the macrophages and use them for dissemination.5 If not destroyed promptly, the microorganisms can replicate in the upper airways of dogs and cats, infect the CNS through the cribriform plate, or colonize parenchymal organs via the bloodstream. Cryptococcus neoformans and C gattii can also grow at temperatures > 30°C, making them able to replicate at physiologic temperatures. Furthermore, these species and a few species within the non–C neoformans group produce a laccase enzyme that catalyzes melanin synthesis. This enzyme aids in protection of the microorganisms from oxidative damage.7

In dogs, hematogenous dissemination of microorganisms is rare and is usually associated with immunosuppression. In the dog of this report, systemic disease was clinically suspected; however, complete necropsy to confirm systemic infection was not performed. Additionally, although yeast organisms were detected in a urine sediment preparation, review of a stained sediment smear would have been necessary to further characterize the organisms. Cryptococcal osteomyelitis has been reported in the literature as part of a systemic condition2 or as a purported primary lesion.8,9 In bone infections, cryptococcal organisms are generally confined to the medullary cavity, where they promote a pyogranulomatous response.10 The microorganisms’ enzymes, including proteinases and phospholipases, and the associated inflammation enhance osteolysis and remodeling. This can involve adjacent bones and can result in necrosis, as evident in the present case. The resulting damaged tissue can provide nutrients for the yeast.6

The diagnosis of cryptococcosis can be made through cytologic examination of samples of lesions, exudates, CSF, and urine sediments from affected patients.1 Romanowsky-type stains, new methylene blue stain, Gram stain, and India ink can be used to enhance detection. Cryptococcal organisms can be also identified in histologic preparations with H&E, periodic acid-Schiff, Gomori methenamine silver, Fontana Masson, Mayer mucicarmine, and Gram stains.2 Cryptococcus spp are extremely variable in size (from 8 to 40 μm in diameter) and can be seen as a smooth form with a thick non-staining capsule or, less frequently, as a nonencapsulated rough form.3 The smaller, poorly encapsulated forms of Cryptococcus spp (Figure 2) may be mistaken for Histoplasma spp. The cytologic distinction may be challenging, but the narrow-based budding characteristic of Cryptococcus spp and the heterogenicity in dimensions of Cryptococcus spp can help to differentiate those 2 microorganisms.4 Serologic evaluation is particularly helpful in speciation of the fungus, given that there are no cytologic features on which to base species identification. Detection of capsular antigen can be done by latex agglutination,1 and fungal culture1 can be performed but is not routinely done to identify this organism.

Dogs with disseminated cryptococcosis have a poor prognosis, and the infection requires prolonged treatment. Antifungal agents such as fluconazole have been used in affected dogs with reasonable success,2 although responses vary. In 1 case, serum titers of C neoformans antigens did not normalize to values expected for a noninfected dog until after 1 year.2 Although cryptococcal osteomyelitis is relatively uncommon, it should be a differential diagnosis in dogs with proliferative bone lesions.

References

  • 1. Malik RKrockenberg MO'Brien CRet al. Cryptococcosis. In: Greene CE, ed. Infectious diseases of the dog and cat 3rd ed. St Louis: Saunders Elsevier, 2006 584598.

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  • 2. Vigiletti APiazza CRusso Vet al. Osteomielite da Cryptococcus neoformans. J Vet (Cremona) 2007 21 16.

  • 3. Cowell RLTyler RDMeinkoth JHet al. Selected infectious agents. In: Cowell RLTyler RDMeinkoth JHet al, eds. Diagnostic cytology and hematology of the dog and cat 3rd ed. St Louis: Mosby Elsevier, 2008 5455 270271.

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  • 4. Lester SJMalik RBartlett KHet al. Cryptococcosis: update and emergence of Cryptococcus gattii. Vet Clin Pathol 2011 40 417.

  • 5. Botts MRHull CM. Dueling in the lung: how Cryptococcus. Curr Opin Microbiol 2010 13 437442.

  • 6. Ma HMay RC. Virulence in Cryptococcus. Adv Appl Microbiol 2009 67 131190.

  • 7. Bovers MHagen FBoekhout T. Diversity of C. neoformans–C. gattii. Rev Iberoam Micol 2008 25 S4S12.

  • 8. Burraco PGallo MG. Localizzazione ossea apparentemente primaria di Cryptococcus neoformans in a dog). J Vet (Cremona) 1988 2 99106.

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  • 9. Brearley MJJeffery N. Cryptococcal osteomyelitis in a dog. J Small Anim Pract 1992 33 601604.

  • 10. Thompson K. Bones and joints. In: Jubb, Kennedy and Palmer's pathology of domestic animals 5th ed. 1 St Louis: Saunders Elsevier, 2007 99100.

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    • Export Citation
  • Figure 1—

    Photomicrograph of a fine-needle aspirate specimen of a proliferative bone lesion at the distal diaphysis of the left humerus of a 4-year-old mixed-breed dog that had had intermittent left forelimb lameness of 3 months’ duration and had suddenly become blind prior to evaluation. Modified Wright stain; bar = 50 μm.

  • Figure 2—

    Photomicrograph of a section of a bone wedge obtained from the affected region of the left humerus of the dog in Figure 1. Notice the severe pyogranulomatous inflammation admixed with cryptococcal yeasts. Bone necrosis and remodeling were also present. H&E stain; bar = 100 μm.

  • Figure 3—

    Photomicrograph of a section of a bone wedge obtained from the affected region of the left humerus of the dog in Figure 1. Cryptococcal yeasts have been phagocytized by macrophages and are also observed in extracellular locations. Many foamy macrophages and fewer neutrophils are present. H&E stain; bar = 50 μm Inset—Cryptococcal organisms with narrow-based budding. H&E stain; bar = 10 μm.

  • 1. Malik RKrockenberg MO'Brien CRet al. Cryptococcosis. In: Greene CE, ed. Infectious diseases of the dog and cat 3rd ed. St Louis: Saunders Elsevier, 2006 584598.

    • Search Google Scholar
    • Export Citation
  • 2. Vigiletti APiazza CRusso Vet al. Osteomielite da Cryptococcus neoformans. J Vet (Cremona) 2007 21 16.

  • 3. Cowell RLTyler RDMeinkoth JHet al. Selected infectious agents. In: Cowell RLTyler RDMeinkoth JHet al, eds. Diagnostic cytology and hematology of the dog and cat 3rd ed. St Louis: Mosby Elsevier, 2008 5455 270271.

    • Search Google Scholar
    • Export Citation
  • 4. Lester SJMalik RBartlett KHet al. Cryptococcosis: update and emergence of Cryptococcus gattii. Vet Clin Pathol 2011 40 417.

  • 5. Botts MRHull CM. Dueling in the lung: how Cryptococcus. Curr Opin Microbiol 2010 13 437442.

  • 6. Ma HMay RC. Virulence in Cryptococcus. Adv Appl Microbiol 2009 67 131190.

  • 7. Bovers MHagen FBoekhout T. Diversity of C. neoformans–C. gattii. Rev Iberoam Micol 2008 25 S4S12.

  • 8. Burraco PGallo MG. Localizzazione ossea apparentemente primaria di Cryptococcus neoformans in a dog). J Vet (Cremona) 1988 2 99106.

    • Search Google Scholar
    • Export Citation
  • 9. Brearley MJJeffery N. Cryptococcal osteomyelitis in a dog. J Small Anim Pract 1992 33 601604.

  • 10. Thompson K. Bones and joints. In: Jubb, Kennedy and Palmer's pathology of domestic animals 5th ed. 1 St Louis: Saunders Elsevier, 2007 99100.

    • Search Google Scholar
    • Export Citation

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