Cryptococcus gattii Type VGIIa Infection in Harbor Seals (Phoca vitulina) in British Columbia, Canada

Since the late 1990s, cryptococcosis has been an emerging zoonotic disease in southwestern British Columbia, Canada (Fyfe et al. 2008). Subsequent to initial detection in humans and veterinary case material on Vancouver Island, British Columbia, the incidence of Cryptococcus gattii infection in western Canada is now among the highest world-wide (Datta et al. 2009). Cryptococcus gattii has been documented in a variety of wildlife and domestic species including dogs (Canis familiaris), cats (Felis catus), horses (Equus caballus), goats (Capra hircus), bottlenose dolphin (Tursiops truncatus), spinner dolphin (Stenella longirostris), Dall's porpoise (Phocoenoides dalli), and harbor porpoise (Phocoena phocoena) (Miller et al. 2002; Stephen et al. 2002; Lester et al. 2004, 2011; Rotstein et al. 2010; Duncan et al. 2011; Norman et al. 2011; Huckabone et al. 2015). There is one report of a California sea lion (Zalophus californianus) with Cryptococcus albidus infection; however, despite intensive surveillance and recovery efforts, no cases of C. gattii have been identified in pinnipeds (Mcleland et al. 2012).

Cryptococcus gattii is one of over 37 members of the genus Cryptococcus within the order Tremellomycetes (Lester et al. 2011). It is a basidiomycetous yeast that, unlike Cryptococcus neoformans, is considered a pathogen of immunocompetent individuals (Sorrell 2001). Until recently, C. gattii was believed to be limited to the tropical and subtropical regions, paralleling the distribution of eucalyptus trees (Sorrell 2001); its emergence in temperate regions has led to numerous collaborative investigations into the extent of environmental contamination, disease transmission, and potential risk of human and animal exposure (Kidd et al. 2007). Exposure to C. gattii can have different clinical manifestations depending on the infected species, route of exposure, inoculum dose, and stage of pathogenesis of infection, but is commonly associated with respiratory and neurologic disease with more-generalized tissue involvement in a subset of animals (Chen et al. 2014). Typical lesions observed with C. gattii in humans include meningoencephalitis, pulmonary cryptococcomas, and cryptococcemia (Chen et al. 2012). In cetaceans, C. gattii infection typically presents as bronchopneumonia (Stephen et al. 2002).

On 16 September 2014, an approximately 3-wk-old female harbor seal (Phoca vitulina) was recovered from a beach in Roberts Creek along the Strait of Georgia, British Columbia, Canada (49°25′12′′N, 123°37′12′′W). The animal was initially assessed and stabilized and then transported to Vancouver Aquarium's Marine Mammal Rescue Centre, Vancouver, British Columbia. The seal weighed 9 kg, was dehydrated, malnourished, and had multiple puncture wounds on the hind flippers. Initial complete blood count and serum chemistry identified a significant leukocytosis (31.2×10⁹/L), neutrophilia (25.6×10⁹/L) with left shift (1.2×10⁹/L bands), transient hyperglycemia (16.1 mmol/L) associated with prior dextrose administration, and age-related changes consistent with published reference ranges (Greig et al. 2010). She was treated supportively with oral fluids (Revibe®, Zoetis Canada, Kirkland, Quebec, Canada) via gastric tube, subcutaneous fluids (Lactated Ringer's, B. Braun Medical Inc., Scarborough, Ontario, Canada), and was started on oral ciprofloxacin (10 mg/kg, 250 mg tablets, Mylan Pharmaceuticals ULC, Etobicoke, Ontario, Canada) and carprofen (2 mg/kg, 25 mg tablets, Zoetis Canada). She remained quiet and rousable but died on the third day post admission. A large volume of mucohemorrhagic discharge was evident from the nares at the time of death.

On 2 April 2015, a second, unrelated animal stranded on a beach in Parksville, British Columbia (49°19′48′′N, 124°16′48′′W). This adult male animal weighed 90 kg, was dehydrated, underconditioned, and minimally responsive to external stimuli. Complete blood count and serum chemistry obtained the day the animal was found identified a moderate leukocytosis (17.3×10⁹/L) and azotemia (blood urea nitrogen [BUN] 15.8 mmol/L; creatinine 107 μmol/L). Repeated blood work showed a persistent leukocytosis (16.1×10⁹/L) with mildly improved azotemia (BUN 13.4 mmol/L; creatinine 75 μmol/L). He was initially treated with subcutaneous ceftiofur (7 mg/kg, 200 mg/mL, Excede 200, Zoetis Canada) and enrofloxacin (5 mg/kg, 50 mg/mL, Baytril®, Bayer Inc., Toronto, Ontario, Canada) in addition to intramuscular dexamethasone (0.25 mg/kg, 5 mg/mL, Dexamethasone5, Vetoquinol, Lavaltrie, Quebec, Canada). The animal passed a large volume of melena, so he was treated with intramuscular duplocillin (1 mL/10 kg, 300,000 IU/mL, Inervet Canada LTD, Whitby, Ontario, Canada). He was treated supportively with subcutaneous fluids (Lactated Ringers, B. Braun Medical) overnight. The following morning, whole body radiographs disclosed patchy pulmonary consolidation, but ultrasound did not detect thoracic effusion. The animal succumbed during a seizure on the second day post admission.

Necropsy and histopathology were consistent in both animals and revealed granulomatous lymphadenitis, meningoencephalitis, bronchopneumonia, and urinary cystitis (Fig. 1). Florid extracellular yeast morphologically consistent with Cryptococcus spp. were identified in associated inflammatory infiltrates in lymph nodes, brain, lung, and the urinary bladder (Fig. 2). The peripheral vasculature had prominent fungemia. Punctate cryptococcomas were observed in the pancreas, salivary gland, tracheal submucosa, and thyroid gland. There were no significant lesions in other tissues examined.

Fresh lung tissue and lymph nodes were submitted for fungal culture on Sabouraud dextrose agar with gentamicin (Oxoid Company, Nepean, Ontario, Canada) media and Columbia blood agar with 5% sheep blood (Oxoid). An isolate of Cryptococcus sp. from each animal was forwarded to the British Columbia Center for Disease Control Public Health Laboratory (Vancouver, British Columbia, Canada) for specific identification and genotyping. Cryptococcus genotyping was performed using restriction fragment length polymorphism PCR methodology as previously described (Kidd et al. 2004, 2005). Briefly, the Ura5 gene fragment was amplified and digested using two sets of restriction enzymes (Set A: Hha and Sau96I and Set B: Hha, BsrG I, and Dde I). Cryptococcus gattii genotypes were determined based on band patterns. The genotyping studies confirmed these isolates as C. gattii VGIIa. Molecular diagnostics to screen for additional pathogens were performed and proved negative for Apicomplexa, Brucella spp., and canine distemper virus with methods previously described (Barrett et al. 1985; Casans et al. 2001; Gibson et al. 2011). Serum antigen titers for Cryptococcus spp. were ≥1,024 for both animals (CrAg® Lateral Flow Assay, Imprimis Pharmaceuticals, San Diego, California, USA). The necropsy findings, histopathology, fungal culture, and serum antibodies confirm that these animals died from disseminated C. gattii VGIIa infection. Based on a review of case records, these are believed to be the first cases of C. gattii in harbor seals or any pinniped species.

With cryptococcosis, clinical findings in affected animals can often vary based on host species and route of exposure (Chen et al. 2014). In terrestrial species, C. gattii may be introduced via inhalation, retrograde invasion via the cribriform plate, ingestion, and direct inoculation by a penetrating wound (Kidd et al. 2007; Pennisi et al. 2013). Based on the distribution and extent of lesions in these harbor seals, the route of exposure was likely via inhalation, and initial infection was presumably in the wild. There were no apparent pre-existing lesions which may have predisposed the animals to infection.

In utero transmission of C. gattii has been documented in a female harbor porpoise (Norman et al. 2011). Although infection of the pup in this case was likely incurred postpartum, the possibility of vertical transmission cannot be entirely discounted based on the age and severity of the lesions. The contribution of insufficient colostrum consumption or other disorders in predisposing this animal to infection is unknown. There has been an association of C. gattii in a Dall's porpoise (Phocoenoides dalli), with identical strains found in terrestrial animals, suggesting a common environmental source of exposure (Huckabone et al. 2015).

The diagnosis of cryptococcosis in these two cases expands the number of susceptible host species and suggests that C. gattii continues to pose a threat to human and animal populations. Fungal culture is recommended in suspected cases and empiric antifungal therapy may be indicated. Ongoing surveillance is recommended to further understand the implications of cryptococcosis for pinniped population health within the northeastern Pacific.

The authors thank all of the staff and volunteers at the Vancouver Aquarium's Marine Mammal Rescue Centre for their dedication and hard work on this patient and all of the other animals in their care. The staff of the Animal Health Center (Abbotsford, British Columbia, Canada) is also recognized for their assistance with the necropsies.