Cryptococcus gattii with bimorphic colony types in a dog in western Oregon: additional evidence for expansion of the Vancouver Island outbreak

Abstract

Cryptococcus gattii was isolated from a 1.5-year-old dog with systemic cryptococcosis in Oregon. The dog had no link to Vancouver Island or British Columbia, Canada. Samples from a nasal swab and from a granulomatous mass within the cranial cavity were pooled for culture. Colonies on Sabouraud dextrose agar were mucoid and exhibited bimorphic morphology, melanin-pigmented and unpigmented. Pigmented colonies were encapsulated budding spherical yeast, whereas unpigmented colonies were of unencapsulated ovoid budding yeast. In addition to defective melanin production, the unpigmented colony type exhibited defective mating. Genetic analysis by high-resolution multilocus sequence typing revealed that the 2 isolates are genetically identical at 8 unlinked loci tested and that the 2 isolates are both the VGIIa Vancouver Island major genotype. Findings are consistent with expansion of the Vancouver Island outbreak onto the mainland Pacific Northwest region of the United States.

Figure 1

Granulomatous mass from the cranial cavity of a 1.5-year-old dog. There are many encapsulated spherical to oval yeast. Hematoxylin and eosin. Bar = 40 μm.

Figure 2

Yeast exhibiting narrow-based budding with intense capsular staining with Mayer’s mucicarmine stain. The organism is surrounded by inflammation consisting of macrophages, lymphocytes, and plasma cells. Mayer’s mucicarmine stain. Bar = 10 μm.

Figure 3

Colonies exhibit bimorphic morphogy on L-dopamine agar. Both are mucoid, with melanin production in the colony on the right, and lack of melanin production in the colony on the left.

Figure 4

Mating differences on Mirashige and Skoog media with myo-inositol, incubated at room temperature in the dark. There is a lack of fertility in the yeast from the colony lacking melanin production, evidenced by lack of filamentous growth (A) whereas filamentous growth indicative of mating is evident in the yeast from the pigmented colony (B). Phase contrast microscopy.

Figure 5

Multilocus sequence typing (MLST) analysis of the isolates with controls, performed for eight unlinked loci. Numbers and color-coding represent different alleles designated by genetic sequence variation, with allele numbers and designations used in this study including GenBank accession numbers (http://209.85.141.104/search?q=cache:lfp_MuMZIN8J:www.nature.com/nature/journal/v437/n7063/extref/nature04220-s7.xls+Genbank+accession+AY973641&hl=en&ct=clnk&cd=1&gl=us&client=firefox-a). Isolate R272 represents the VGIIb minor outbreak genotype control, isolate R265 represents the VGIIa major outbreak genotype control, and isolates WM276, WA 861, NIH312, and MMRL2651 represent the VGI-VGIV controls for each molecular group respectively. Isolates EJB17a and EJB17b from the dog in this report each share identical genotypic profiles with R265 at these 8 loci, indicating they are VGIIa outbreak genotype isolates.