The zoo says three-year-old Juntos died Tuesday night despite feverish attempts by animal care workers to save the animal.
Another three-year-old named Guillermo died on Monday.
Both penguins came to the Calgary Zoo from an animal park in Syracuse, N.Y.
The Humboldts make their summer home in the outdoor exhibit at the popular Penguin Plunge.
It was one of the first exhibits to reopen two weeks ago after severe flooding damaged much of the zoo in June.
The zoo has three other species of penguin, but so far only the Humboldts appear to have fallen ill.
“I have conducted the post-mortem examinations on both penguins and, while we are waiting for confirmation, the results are most consistent with an infection caused by the bacterium Chlamydophila psittaci," Dr. Doug Whiteside, the zoo's senior staff veterinarian, said in a release Wednesday.
“Test results will confirm this diagnosis, but in the meantime we are treating the remaining eight Humboldts and two gentoos that share the outdoor exhibit to do our best to prevent further losses.”
Whiteside said the disease has caused outbreaks in other zoos and has been detected in wild Humboldts and other bird species. It can be latent for months or years.
“All Humboldts have just completed their annual molt where they naturally shed all their feathers over a period of two to three weeks,” explained Whiteside. “During this time of physiological stress the birds can lose as much as 20 per cent of their body weight and can be more susceptible to infections.”
Other penguin species at the zoo include kings and rockhoppers.
Humboldt penguins are native to Chile and Peru. They are named after the cold water current they swim in and which is itself named after geographer, naturalist and explorer Alexander von Humboldt.
The penguins are medium in size and weigh up to six kilograms. They have a black head with a white border that runs from behind the eye, around the ears and chin.
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