Introduction

A 25-year-old 4.4-kg male aquarium-hatched African penguin (Spheniscus demersus) was evaluated because of a raised pigmented mass that measured 1.5 × 0.5 cm and originated from within the right naris that was noted 2 days prior. The penguin had been apparently healthy except for bilateral cataracts and intermittent lameness of the hind limbs. The penguin received supplemental nutraceuticals (250 mg of glucosamine hydrochloride, 200 mg of sodium chondroitin sulfate, and 2 mg of manganese, PO, q 24 h). The penguin was 1 of 31 African penguins housed at an aquarium in a 32,000-gallon outdoor freshwater habitat with adjacent dry resting areas. At the time of presentation, the penguin was bright and alert with normal appetite and behavior; physical examination findings were unremarkable except for the pigmented mass from the right naris. Examination of a fine-needle aspirate specimen collected at the time of presentation revealed a large number of uniform cells, each with a large round nucleus and variable darkly pigmented cytoplasmic granules (Figure 1). The presumptive diagnosis was malignant melanoma, and an excisional biopsy procedure was performed. The penguin was anesthetized with sevoflurane, and the mass and surrounding beak were scrubbed with diluted 10% chlorhexidine solution and isopropyl alcohol. The external portion of the pigmented mass was excised with No. 15 scalpel blade; however, it was noted to extend into the nasal cavity. A No. 15 scalpel blade and a bone curette were used to remove as much of the visibly pigmented mass from the nasal cavity as possible, but clean surgical margins could not be achieved. Hemostasis was achieved by application of gel-foam (Pfizer), and the penguin recovered uneventfully from anesthesia. A blood sample (6.0 mL) was collected from the right jugular vein with a 21-gauge butterfly needle and syringe for a CBC and plasma biochemical profile. Clinicopathologic findings were considered normal for this species. Histologic examination of sections of the pigmented mass revealed a well-demarcated but unencapsulated, expansile, infiltrative, densely cellular neoplasm, which was confirmed to be a malignant melanoma (Figure 2). In the examined tissue sections, there were 0 to 2 mitotic figures/hpf (100X).

Photomicrograph of a fine-needle aspirate specimen from a raised 1.5 × 0.5-cm pigmented mass extending from within the right naris of a male African penguin (Spheniscus demersus) that was noted 2 days earlier. Notice a large number of uniform cells each with a large round nucleus (asterisk) and variable darkly pigmented cytoplasmic granules (dagger). Romanowsky stain; bar = 10 μm.

Citation: Journal of the American Veterinary Medical Association 260, 4; 10.2460/javma.20.10.0564

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Photomicrograph of a fine-needle aspirate specimen from a raised 1.5 × 0.5-cm pigmented mass extending from within the right naris of a male African penguin (Spheniscus demersus) that was noted 2 days earlier. Notice a large number of uniform cells each with a large round nucleus (asterisk) and variable darkly pigmented cytoplasmic granules (dagger). Romanowsky stain; bar = 10 μm.

Citation: Journal of the American Veterinary Medical Association 260, 4; 10.2460/javma.20.10.0564

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Photomicrograph of a fine-needle aspirate specimen from a raised 1.5 × 0.5-cm pigmented mass extending from within the right naris of a male African penguin (Spheniscus demersus) that was noted 2 days earlier. Notice a large number of uniform cells each with a large round nucleus (asterisk) and variable darkly pigmented cytoplasmic granules (dagger). Romanowsky stain; bar = 10 μm.

Citation: Journal of the American Veterinary Medical Association 260, 4; 10.2460/javma.20.10.0564

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Photomicrograph of a section of the pigmented mass surgically removed from penguin’s upper beak. The heavily pigmented mass extends out from the right naris and onto the upper beak (asterisk) and has invaded and replaced skin, bone, and connective tissue. The mass abuts the cartilage of the nasal cavity (dagger) and also has invaded some areas. H&E stain; bar =100 μm. The mass was diagnosed as a malignant melanoma.

Citation: Journal of the American Veterinary Medical Association 260, 4; 10.2460/javma.20.10.0564

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Photomicrograph of a section of the pigmented mass surgically removed from penguin’s upper beak. The heavily pigmented mass extends out from the right naris and onto the upper beak (asterisk) and has invaded and replaced skin, bone, and connective tissue. The mass abuts the cartilage of the nasal cavity (dagger) and also has invaded some areas. H&E stain; bar =100 μm. The mass was diagnosed as a malignant melanoma.

Citation: Journal of the American Veterinary Medical Association 260, 4; 10.2460/javma.20.10.0564

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Photomicrograph of a section of the pigmented mass surgically removed from penguin’s upper beak. The heavily pigmented mass extends out from the right naris and onto the upper beak (asterisk) and has invaded and replaced skin, bone, and connective tissue. The mass abuts the cartilage of the nasal cavity (dagger) and also has invaded some areas. H&E stain; bar =100 μm. The mass was diagnosed as a malignant melanoma.

Citation: Journal of the American Veterinary Medical Association 260, 4; 10.2460/javma.20.10.0564

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Histologic examination confirmed extension of the neoplastic tissue to the lateral margins of the tissue sections; thus, local tumor recurrence and metastasis were concerns. Because the mass had been excised without clean margins, adjunctive treatments, such as oral administration of antitumor drugs, radiation therapy, and immunotherapy were considered to reduce the likelihood of neoplastic recurrence or metastasis. Treatment of the penguin with meloxicam (0.2 mg/kg, PO, q 24 h) was commenced and continued for a total of 45 weeks; however, the treatment was discontinued for a period of 6 weeks because of the risk of toxic effects in a chick that the penguin was feeding.

Five weeks after the excisional biopsy was performed, the penguin underwent local hypofractionated radiation therapy with a single 9 MeV electron beam. For each radiation procedure, the penguin was anesthetized by mask induction with isoflurane and oxygen and subsequent intubation. The penguin received 4 once weekly fractions of radiation (8 Gy/fraction) for a total dose of 32 Gy. During each treatment, bolus material (1.0 cm thick; soft tissue equivalent) was placed over the irradiation field to ensure dose homogeneity.

For a multimodal approach to malignant melanoma management, immunotherapy was pursued, and the penguin was administered a commercially available canine melanoma vaccine (Oncept; Merial Ltd). The first vaccination was given 5 weeks after the excisional biopsy, at the same time as the first radiation therapy treatment. The vaccination schedule involved an initial 4-dose series, with 1 injection given every other week. Each dose was delivered by use of the transdermal administration device provided by the manufacturer, which uses a spring action propeller system to deliver the vaccine dose through the skin and into the underlying tissue. After this initial series, a booster vaccination was administered 7 months after the first dose. No adverse effects during or after vaccine administration were noted.

Before, during, and after vaccine administration, the penguin’s anti–human tyrosinase (HuTyr)-specific antibody titer was evaluated. A blood sample was collected from the penguin before the vaccination protocol was commenced (day –3), prior to the first dose administration (day 0), immediately prior to each subsequent vaccine administration (days 14, 28, and 42) and booster vaccine administration (day 224), and at a scheduled vaccination recheck (day 280). At each time point, approximately 6 mL of blood was collected from the penguin’s right jugular vein with a 21-gauge butterfly needle and a 6-mL syringe and placed into serum separator tubes. Serum was separated by centrifugation at 1,200 × g and stored at –80 °C until analyzed.

A standard ELISA for anti-HuTyr antibody detection was used according to published methods^1,2 to determine the penguin’s tyrosine-specific antibody titer at the various time points. A microtiter plate (Immulon; Thermo Fisher Scientific) was coated overnight (approx 12 hours) at 4 °C with full-length recombinant HuTyr (Human tyrosinase protein; Abnova Corporation; 50 μL/well) and then washed with PBS-Tween (0.05%) solution. A mixture of 2% milk and PBS solution (150 μL/well) was then added to each well to reduce nonspecific antigen binding, and after 1 hour of incubation at 37 °C, the plates were again washed. Aliquots of the penguin’s serum samples (50 μL/well) were then added and incubated for 1 hour at 37 °C, after which the plates were washed. A secondary horseradish peroxidase-conjugated goat anti-chicken IgY antibody (anti-chicken immunoglobulin Y [IgY] antibody; Sigma-Aldrich Laboratories) was added (50 μL of a 1:30,000 dilution/well) and incubated for 1 hour; the plates were washed and then developed with 100 μL of a 3,3′,5,5′-tetramethylbenzidine substrate solution (Tetramethylbenzidine [TMB] substrate solution; Thermo Fisher Scientific). Reactions were stopped after approximately 5 to 10 minutes by adding 50 μL of 0.18M H²SO⁴/well. The optical density at 450 nm (OD450) values were measured with a microplate reader instrument (ELx800^®; Bio-Tek Instruments). Human glutathione S-transferase protein (Human GST protein; Abnova Corp) was used as a negative control. Reactions were run in triplicate, and the mean OD450 value was recorded for each time point.

The penguin’s antiHuTyr-specific antibody titer increased during vaccination protocol’s core period (days 0 to 42). The serum antibody titer at day 224 (7 months after the first dose) was below measurable values. A booster injection was administered to the penguin on day 224 (7 months after the first dose); 2 months later, there was a dramatic rebound in the serum OD450 value (the highest postvaccination anti–HuTyr-specific antibody titer [Figure 3]). Overall, there was a 3-fold increase in the penguin’s anti–HuTyr-specific antibody titer, indicating a positive humoral immune response to the canine melanoma vaccine.

Humoral (IgY) immune response in an African penguin (Spheniscus demersus) before and after treatment with a canine melanoma vaccine (a xenogenic DNA vaccine encoding anti–human tyrosinase), which elicits an antigen-specific immune response in the host that is directed against melanoma cells. The first vaccine dose was administered on day 0. The vaccination schedule involved an initial 4-dose series, with 1 transdermal injection given every other week. A booster vaccination was administered on day 224. Humoral response was assessed as optical density measurements obtained with a microplate reader with 450-nm filter (optical density at 450-nm values).

Citation: Journal of the American Veterinary Medical Association 260, 4; 10.2460/javma.20.10.0564

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Humoral (IgY) immune response in an African penguin (Spheniscus demersus) before and after treatment with a canine melanoma vaccine (a xenogenic DNA vaccine encoding anti–human tyrosinase), which elicits an antigen-specific immune response in the host that is directed against melanoma cells. The first vaccine dose was administered on day 0. The vaccination schedule involved an initial 4-dose series, with 1 transdermal injection given every other week. A booster vaccination was administered on day 224. Humoral response was assessed as optical density measurements obtained with a microplate reader with 450-nm filter (optical density at 450-nm values).

Citation: Journal of the American Veterinary Medical Association 260, 4; 10.2460/javma.20.10.0564

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Humoral (IgY) immune response in an African penguin (Spheniscus demersus) before and after treatment with a canine melanoma vaccine (a xenogenic DNA vaccine encoding anti–human tyrosinase), which elicits an antigen-specific immune response in the host that is directed against melanoma cells. The first vaccine dose was administered on day 0. The vaccination schedule involved an initial 4-dose series, with 1 transdermal injection given every other week. A booster vaccination was administered on day 224. Humoral response was assessed as optical density measurements obtained with a microplate reader with 450-nm filter (optical density at 450-nm values).

Citation: Journal of the American Veterinary Medical Association 260, 4; 10.2460/javma.20.10.0564

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During the subsequent several months, the penguin was monitored by weekly physical examinations, including a CBC and plasma biochemical profile, and no abnormalities were identified. Eight months after the initial presentation, there was recurrence of tumor growth at the original site (0.3 × 0.2 cm). A fine-needle aspirate specimen was collected, the examination of which confirmed the diagnosis of melanoma. At this time, local radiation therapy was repeated. The penguin was anesthetized, and local radiation therapy involving strontium-90 was applied with a 4-mm-diameter circle probe in nonoverlapping circles (dose of 75 Gy) over a period of 11.5 minutes. One week later, the penguin was again anesthetized and retreated with an equivalent dose of radiation. The penguin continued to do well during the following several weeks, with no concerns reported by the aquarium staff. At 11 months after the initial presentation, the melanoma had increased in size (0.5 × 0.3 cm) and was more fluctuant on palpation. The penguin had noticeable weight loss (body weight, 3.1 kg [4.4 kg at the time of diagnosis]), regurgitation, lethargy, and moderate to severe leukocytosis. The leukocytosis continued for several weeks (WBC count ranged from 27,300 to 63,300 cells/μL; reference range, 16,000 to 26,100 cells/μL) and was characterized by heterophilia (heterophil count ranged from 16,571 to 48,108 cells/μL; reference range, 8,853 to 14,791 heterophils/μL), lymphocytosis (lymphocyte count ranged from 3,814 to 8,862 lymphocytes/μL; reference range, 5,217 to 11,903 cells/μL), and monocytosis (monocyte count ranged from 1,053 to 5,280 monocytes/μL; reference range, 119 to 535 monocytes/μL). The penguin was treated with gastrointestinal protectants including sucralfate (500 mg, PO, q 12 h) and famotidine (0.5 mg/kg, PO, q 12 h) and metoclopramide as an anti-emetic (0.5 mg/kg, PO, q 12 h); itraconazole (5 mg/kg, PO, q 12 h) and ceftiofur crystalline-free acid (14 mg/kg, IM, q 72 h) were also administered. The penguin received fluid therapy with lactated Ringer solution (15 mL/kg, SC, q 24 h) and tube feedings of fish gruel (15 to 30 mL, q 8 h). A whole-body CT scan performed with contrast agent administration 1 month later revealed no apparent evidence of metastatic neoplasia or pulmonary disease. The bird’s condition continued to decline over the next few weeks, and it died at 15 months after initial presentation. Necropsy and histologic examination revealed malignant melanoma of the upper beak and aspergillosis of the lungs and air sacs. There was no evidence of metastasis from the malignant melanoma.

Discussion

Penguins are a popular display species in zoos and aquariums worldwide. The African penguin (Spheniscus demersus) is endemic to southern Africa and is listed as endangered under the International Union for Conservation of Nature’s Red List of Threatened Species because of a population decrease > 50% in the 3 most recent generations.^3,4 As free-ranging populations continue to decrease, it is important to continue to maintain these birds within zoos and aquariums, where they have a role in educating the public with regard to the species’ biology and conservation needs. In addition, it is critically important to continue to investigate new techniques in medical diagnostic testing and treatments for animals under managed captivity to ensure their health and longevity.

Malignant melanomas have been reported for several avian species including a lesser black-backed gull (Larus fuscus), a thick-billed parrot (Rhynchopsitta pachyrhyncha), a merlin (Falco columbarius), a mandarin duck (Aix galericulata), a zebra finch (Taeniopygia guttata), and a red-tailed hawk (Buteo jamaicensis).^5,6,7,8,9,10 Malignant melanomas in macaroni (Eudyptes chrysolophus), rock hopper (Eidyptes chrysocome), Humboldt (Spheniscus humboldti), and African penguins have also been reported.^11,12 In penguins, these aggressive neoplasms typically develop on the nonfeathered skin of the feet or tibiotarsal-tarsometatasal joint (hock), subcutaneous muscle, beak or cere, and the skin of the inguinal region with a reported prevalence of 2%.¹¹ Preliminary diagnosis is typically made by histologic examination of fine-needle aspirate specimens, and definitive diagnosis is made by cytologic examination of tissue biopsy specimens.¹¹ Malignant melanomas tend to recur and have a high rate of metastasis, regardless of affected species.^5,9,10 Malignant melanomas have been reported to spread to the liver, lungs, adrenal glands, brain, and bones in several avian species.^{5,8,9,10,11,12} Results of 1 study¹¹ indicated that in macaroni (n = 5), rock hopper (3), and Humboldt (2) penguins, tumors ≥ 2.0 cm diameter with ulcerated surfaces were likely to have metastasized. On the basis of this information, it was suggested that penguins with tumors < 2.0 cm diameter that undergo aggressive mass resection may have a lower risk of metastasis.¹¹ In the same study, the mean survival time after diagnosis in those 3 species of penguins was 7 months.¹¹

The penguin of the present report had a raised pigmented mass (measuring < 2 cm) on the maxillary beak near the nares. This is a common site for the development of melanoma in penguins.¹¹ The primary mass was surgically removed within 2 weeks after presentation; however, it was not possible to excise the tumor with clean margins due to the location of the mass and the lack of available tissue with which to obtain wide surgical margins. For this reason, a multimodal approach to long-term tumor management was warranted. The penguin received meloxicam (a cyclooxygenase-2 [COX] inhibitor), which is an NSAID that has been suggested to act as a cancer chemopreventative agent.¹³ In addition, the penguin received local hypofractionated radiation treatments at a dose of 8 Gy/fraction (total radiation dose, 32 Gy) with no notable adverse effects. Immunotherapy was also pursued, and the penguin was vaccinated with a canine melanoma vaccine, with no apparent adverse effects.

Current treatment recommendations for patients with malignant melanoma include complete surgical excision with or without radiation therapy and systemic adjunct treatment.¹⁴ Complete surgical excision presents a challenge in penguins and other avian species, especially at sites such as the beak or oral cavity, because of the limited soft tissues available to obtain clean surgical margins. For this reason, adjunct treatments such as radiation therapy and orally administered antitumor drugs are often used. Orally administered antitumor drugs, such as the COX-2 inhibitors meloxicam and piroxicam, block tumor growth by antiangiogenic and proapoptotic effects.¹⁵ Cyclooxygenase is an enzyme that regulates many cell functions, including prostaglandin production.¹⁶ The inhibition of prostaglandin production can negatively affect cell proliferation and tumor growth.¹⁶ Adverse effects of long-term use of the COX-2 inhibitors in humans include gastric mucosal erosions that can lead to gastric ulceration, bleeding, and perforation.¹⁶ Radiation therapy for melanoma treatment typically consists of hypofractionated radiation therapy, which differs from conventional radiation therapy by the division of the total cumulative dose into relatively large individual treatments that may be administered daily or less frequently.¹⁷ This targeted radiation therapy, which requires patient transport to a radiation oncology facility and immobilization with general anesthesia, is used to improve local control of the cancer and may also provide analgesia.

Immunotherapy is a type of biological treatment that either stimulates or suppresses the immune system to help the body fight cancer, infection, or other diseases. The ideal cancer immunotherapy agent should have high specificity (the ability to discriminate between cancerous cells and noncancerous cells), high sensitivity (the ability to kill small or large numbers of tumor cells), and high durability (the ability to prevent recurrence of tumor growth).¹⁸ The primary goal of a cancer vaccine is the ability to elicit an antitumor immune response that results in clinical regression of the tumor or reduction in the likelihood of metastasis. It may take several months of treatment before a clinical response to cancer vaccines is evident because of the slow rate of induction of the adaptive immune system.¹⁹ Immunotherapy has been used in domestic mammals with good success, and use in nondomestic animals continues to be investigated.^20,21

The canine melanoma vaccine (Oncept canine vaccine, DNA; Merial Ltd) given to the penguin of the present report was a xenogenic DNA vaccine encoding HuTyr, which elicits an antigen-specific immune response that is directed against melanoma cells in the host. Although the product is labeled for use in dogs, extralabel use in other species including cats has been reported.²² The vaccine targets the protein tyrosinase, which is normally expressed in melanocytes, widely conserved across species, and known to be present in birds.²³ The vaccination regimen involves intradermal injection of a vaccine dose given every other week for 4 doses, followed thereafter by a booster injection every 6 months. Use of the canine melanoma vaccine in nondomestic animals, including a lion with a dermal melanoma, a tiger with a conjunctival fornix melanoma, and an African penguin with a melanoma of the uropygial gland, has been described.²¹ The lion received a full course of the vaccine and radiation therapy, and there was no physical evidence of disease at 6 months after diagnosis. The penguin received 2 vaccine doses prior to euthanasia because of quality-of-life concerns; postmortem examination of the bird revealed evidence of metastasis. No adverse reactions were noted secondary to administration of the melanoma vaccine in any of those cases.²¹

In the case described in the present report, a HuTyr-specific ELISA was used to measure anti-HuTyr antibody titer at various time points in an African penguin. It has been confirmed that commercially available antibodies cross react with immunoglobulins from numerous avian species; therefore, the secondary antibody used in this assay was a horseradish peroxidase-conjugated goat anti-chicken immunoglobulin.²⁴ As highlighted by the findings for the bird of the present report, it was evident that the xenogeneic plasmid DNA vaccine encoding HuTyr can induce an antigen-specific humoral immune response in an African penguin. This penguin mounted a positive humoral immune response to the canine melanoma vaccine, which has been shown in other species to facilitate clinical regression of the tumor and prevention of metastasis.¹⁹ A 3-fold increase in the titer of anti–HuTyr-specific antibodies in the African penguin was identified during the vaccination period (from day 0 to day 42), which is similar to the response reported for dogs.^1,25 Prior to the booster vaccination at 7 months after the first dose, the titer of anti–HuTyr-specific antibodies titers had decreased to below measurable levels. However, at a recheck examination 2 months after the booster vaccination, the titer had rebounded and was greater than that achieved after completion of the initial vaccination protocol, a finding that is similar across most species.^1,2 It is unknown whether penguins should be booster vaccinated earlier than 6 months after the end of the initial vaccination protocol to prevent titers from decreasing to below measurable levels, and further research is needed to determine whether a shorter interval is indicated. These findings are similar those reported for dogs vaccinated with the melanoma vaccine, suggesting the induction of antibody, T-cell, and anti-tumor responses in this penguin.^1,25

It is important to note that some features of the case management could have contributed to the local tumor regrowth in the penguin of the present report. The penguin was started on long-term oral treatment with meloxicam (an NSAID) for the drug’s antitumor effects, and administration of the drug continued for a total of 45 weeks; however, this medication was discontinued for a period of 6 weeks while the bird was on the nest feeding its chick. It is possible that the period of treatment discontinuation may have affected the regrowth of the tumor, because tumor recurrence was noted after the penguin had not received meloxicam for 6 weeks. The COX-2 inhibitors block cyclooxygenase, which regulates prostaglandin production, and the inhibition of prostaglandin production may have an effect on cell proliferation and tumor growth.¹⁶ The NSAID dosage (0.2 mg/kg, PO, q 24 h) given to the penguin was extrapolated from recommendations for cancer prevention in dogs. However, results of a recent study²⁶ that investigated the pharmacokinetics of a single oral or IM dose of meloxicam in African penguins suggested that treatment with meloxicam at 1 mg/kg, PO, every 48 hours or 0.5 mg/kg, IM, every 24 hours was appropriate. It is currently unknown whether an alteration in NSAID dosage would have affected tumor regrowth in the penguin of the present report. Although the adverse effects of long-term use of the COX-2 inhibitors are reported to include gastric mucosal erosions, ulceration, and bleeding or perforation,¹⁰ there were no apparent adverse effects in the penguin based on the clinical and necropsy findings. This African penguin received the booster injection of the melanoma vaccine 6 weeks later than what is typically recommended for canine patients; therefore, this delay may have been a contributing factor in the regrowth of the mass that occurred 8 months after initial diagnosis. It is possible that long-term and consistent administration of meloxicam and proper timing of the melanoma booster vaccination may aid in better control of melanoma growth in this species. It is also important to note that adaptive immunity may have an important role in melanoma pathogenesis, although this was not determined in the case described in the present report.

The African penguin of the present report died as a result of pulmonary and air sac aspergillosis 15 months after the time of the initial diagnosis of malignant melanoma. No evidence of metastasis was identified during necropsy, suggesting a positive clinical response to treatment. Despite this bird’s regular desensitization to handling and travel, it is currently unknown whether repeated off-site transports for specialty diagnostic testing and treatments may have increased the penguin’s risk of developing aspergillosis. Prophylactic treatment with an antifungal during the course of radiation and vaccination could be considered in African penguins, if clinically warranted, to prevent development of aspergillosis. The survival time for this penguin exceeded the mean survival time (7 months) of penguins with malignant melanoma,¹¹ suggesting that multimodal treatment including radiation, melanoma vaccination, and oral administration of antitumor drugs was efficacious for control of disease progression and prevention of metastasis. The notable humoral response 8 weeks after administration of the booster vaccine may have aided in reducing the likelihood of metastasis in this case.

The case described in the present report has suggested that a canine melanoma vaccine (Oncept; Merial Ltd) can be used safely in African penguins and that this species mounts a positive humoral immune response to the vaccine, similar to the postvaccination response recorded for dogs and horses.^1,2 Given the pattern of changes in humoral immunity for this penguin, the vaccine booster appears to be important for adequate antibody rebound effects, similar to findings in other species.^1,2 For improved successful management of malignant melanoma in penguins, immediate implementation of multimodal treatment appears important, although additional research is warranted to further understand how these treatments affect the course of disease. Clinicians who treat penguins with malignant melanoma should consider including oral chemotherapy for the inhibition of prostaglandin production, radiation to decrease the size of the tumor mass locally, and immunotherapy to elicit an antigen-specific immune response directed against melanoma cells in the host.