CRYPTOCOCCOSIS IN STRANDED MARINE

MAMMALS IN BRITISH COLUMBIA

Martin Haulena, Justin Rosenberg, Muhammad

Morshed, Linda Hoang, Erin Zabek, Chelsea

Himsworth, Chelsea Anderson, Lindsaye Akhurst,

Marina Ivančić, Dean A. Malpas, John S. Pollock, and

Stephen Raverty

Vancouver Aquarium

British Columbia Provincial Animal Health Center

AquaVetRad

British Columbia Centre for Disease Control

Canada Diagnostic Centre

Cryptococcus gattii

• Since 2000, there has been a recognized outbreak of cryptococcosis in human and companion animals on Vancouver Island, BC

– From early 1999 to mid 2000, microbiologist on Vancouver Island identified 19 cases of cryptococcosis in humans.

• Most individuals had no evidence of immunosuppression

– Central Laboratory for Veterinarians, Langley, BC reported 12 cases of cryptococcosis in companion animals on Vancouver Island for 2000

• Isolates typed as var gatti type B

Case Summary Information• Porpoises presenting with

cryptococcosis

• Species– 10 Dall’s porpoises (Phocoenoides

dalli)

– 14 harbor porpoises (Phocoena phocoena)

– 1 Pacific white sided dolphin (Lagenorhynchus obliquidens)

• Gender– 10 females

– 11 males• 4 animals unknown

• Age– 20 adults

– 4 juveniles

– 1 fetus• Important implications for humans

and companion animal health

• Venereal or hematogenous mechanisms

Photo: Dr S Norman

Photo: Dr A Hall

Pneumonia

Lymphadenopathy

Ancillary Laboratory Results

• Fungal culture

– Consistently isolated Cryptococcus gatti, type B from lung and mediastinal lymph nodes

– PCR-fingerprinting, RFLP

• Isolates identified as genotype VGII

• Only 2 isolates VGI

– Human fatality

– Dall’s porpoise from West Vancouver, mixed VG1/VG2

Ancillary Laboratory Results• For each submission

– Pooled lung, lymph node, spleen, and brain• Negative for morbillivirus and Brucella spp by polymerase chain reaction

(PCR)

– Fecal floatation identified light to moderate burden of gastrointestinal nematodes

– Seven of 25 liver samples with no detectable vitamin A, 3 with moderately reduced values

– Retinyl palmitate and retinol

– Reference range 300-1200 mcg/gm dry weight

– Immunohistochemistry on select cases• Antibodies available for Cryptococcus spp capsular staining, but not as specific

as culture or molecular studies

• To date, no consistent pre-existing or underlying diseases detected within examined porpoises– Contaminant studies currently underway

Porpoises at VAMMR 2008 to present

• 6 live-stranded

• 6 weeks to adult

– Bimodal stranding pattern

with peaks in early spring

and late summer

• 12.1 to 49.5 kg at admit

• Each animal received 24-

hour care by veterinary staff

and rescue volunteers.

Theo

Theo

Theo

Theo

Theo

Theo

Theo

Theo

• 3 year-old, 41 kg, male sub-adult live

stranded Pacific harbor porpoise (P.

phocoena) admitted July 2012

• Poor body condition, moderate lethargy,

marked weakness, and inability to swim

• moderate dehydration, mucopurulent

discharge from the blowhole, blepharospasm

OU and multiple superficial to deep

necrotizing cutaneous wounds

Theo

• Elevated liver enzymes and mild inflammation

• Fecal parasitology identified Nasitrema sp. and

Stenurus sp. ova and larvae

• Differentials for the clinical signs included peri-

stranding trauma, septicemia, verminous pneumonia

and malnutrition

• Therapy consisted of antibiotics, corticosteroids,

benzodiazepines, antiemetics, bronchodilators,

fenbendazole, praziquantel, rehydration and wound

care

Theo

• 30 days after admission, marked

inflammation was noted

• Yeast was found associated with skin wounds

as well as in gastric samples

• Itraconazole started and inflammation

resolved over the next 16 days and porpoise

appeared better

Theo

• HOWEVER, 58 days after admission, the

porpoise began to slowly deteriorate showing

increased vomiting, weakness and decreased

mobility

• Developed marked scoliosis, head pressing

and erratic head movements

• Radiographs, sonography, and gastroscopy

revealed no significant lesions

The Shanie Factor

• High-field (1.5T) whole-body magnetic resonance imaging (MR)

was performed on Day 71

• MR revealed patchy, coalescing, solid intra-axial brain lesions in

the basal ganglia (caudate nucleus, putamen) and thalamus that

were hyperintense on T2w and FLAIR sequences

• Due to ongoing deterioration and poor prognosis, the porpoise was

euthanized

Cryptococcus testing

• Latex agglutination for cryptococcal antigen

• Ante mortem at Idexx

– NEGATIVE

• Post mortem hindsight at human hospital

lab

– 1:8

– POSITIVE

Conclusions

• First clinical presentations of a primarily neurologic form

of cryptococcosis in a cetacean

•

• First antemortem detection of imaging findings consistent

with central nervous system cryptococcosis

• These findings highlight the need for more rapid

diagnosis to provide better case management for stranded

cetaceans.

And other marine mammals ?

• Since 2014, cryptococcosis has been diagnosed

in two live-stranded harbour seals admitted to the

VAMMR

• A female 3 week old pup and an unrelated adult

male

Clinical presentation

• Pup was malnourished, dehydrated and had

multiple puncture wounds

– Significant leukocytosis

– Died 3 days after admit with mucohemorrhagic nasal

discharge

• Adult was malnourished and dehydrated and

poorly responsive

– Moderate leukocytosis

– Died on second day after admit during a prolonged

seizure

Necropsy results

• Consistent in both animals

– Granulomatous lymphadenitis

– Menigoencephalitis

– Bronchopneumonia

– Urinary cystitis

Histopathology

• Extracellular yeast consistent with

Cryptococcus spp.

– Lymph nodes

– Brain

– Lung

– Urinary bladder

• Peripheral fungemia

• Cryptococcomas in the pancreas, salivary

gland, trachea, and thyroid gland

Localized meningoencephalitis

with focal effacement of the

molecular layer of the

cerebellum and peripheral

displacement and compression

of the adjoining granular layer

Cerebellar necrosis and

associated inflammatory

infiltrate and intralesional yeast.

Lung with varying degrees of

atelectasis and congestion as well

as distended alveoli, occluded by

a myriad of yeast and abundant

mucus

Lymph node involvement with

expansion and replacement of the

cortex by nodular to diffuse

accumulations of yeast with mild

to moderate granulomatous

inflammatory infiltrate

Additional diagnostics

• Genotyping studies confirmed isolates as C.

gattii VGIIa

• Serum antigen titres for Cryptococcus spp.

were > 1,024 fro both animals

– CrAg Lateral Flow Assay, Imprimis

Pharmaceuticals

Acknowledgements

• Vancouver Aquarium’s Marine

Mammal Rescue Centre

• Shanie Fradette

• The marine mammal team

• The amazing vet techs: Chelsea

DeColle and Gwyneth Nordstrom

• The incredible rescue team: Kate

Cooper, Taryn Roberts, Sion

Cahoon

• > 80 volunteers and interns for

their invaluable efforts

• And a whole lot of support from

the whole Vancouver Aquarium

team