Amyloidosis is defined as the deposition of soluble proteins or their fragments extracellularly in various organs. Amyloidosis represents a wide spectrum of protein-misfolding disorders in human and animals (2,8,9). Various proteins that are soluble under physiological conditions can undergo conformational changes to the beta-sheet–rich structure and subsequently self-assemble into highly insoluble amyloid fibrils (8). In human medicine, at least 28 different proteins are known to have the ability to aggregate, insolubilize, and deposit in various tissue as amyloid (8). The amyloid light chain (AL) amyloid type, which is derived from immunoglobulin light chains of plasma cells, is the most common form in humans (12). In animals, at least eight different amyloid precursors have been described, and the AA form of amyloid is the one that is most frequently found. AA amyloid is derived from an acute phase protein called serum amyloid A (SAA) that normally plays a role in cholesterol transport and as a chemoattractant in the inflammatory process (7,19).

Amyloidosis can be divided into 2 major classes such as localized and systemic (generalized) (8,19). In localized amyloidosis in animals, amyloid fibrils are deposited in the brain and the pancreas, in which precursor proteins are synthesized. On the other hand, in systemic amyloidosis, various serum precursor proteins in the blood form amyloid fibrils and then deposit in many organs in body (8,19). The systemic amyloidosis can be further sub-classified as primary amyloidosis (AL) associated with immunocyte dyscrasia, or secondary amyloidosis (AA) associated with a chronic inflammation or destructive tissue process (19). AA amyloidosis is a kind of systemic amyloidosis and occurs in domestic, laboratory, and wild animals with chronic inflammation (8). Avian amyloidosis was first described in a pheasant in 1867 (4). According to previous literatures, this frequently affects waterfowl (Anseriformes, Gruiformes, Phoenicopteriformes); and ducks (Anatidae) seem particularly prone to this condition (16,18). Amyloidosis has also been reported in domestic poultry, captive birds in zoo and parks, and free-living birds (16). Systemic amyloidosis has long been recognized in captive animals, including birds (4). Marine and coastal birds in captivity, especially those of the orders Anseriformes, Gruiformes and Charadriiformes (to which genus Larus belongs) seem to be particularly prone to develop amyloidosis. Therefore, captivity seems to be an apparent risk in birds and over-crowding of animals may be one contributing factor to develop this disease (6).

Several cases of systemic amyloidosis were reported in dog and bird (whopper swan) in Korea (11,18). Here we describe the systemic amyloidosis in an African penguin (Spheniscus demersus) reared in aquarium.

A female, 14-year-old, African penguin (Spheniscus demersus) weighing 2.5 kg with the anorexia for one day and 22 months history of bumblefoot was found dead in aquarium on 21 August 2019. Pododermatitis was observed at the ventral surface of the right foot on 30 November 2017. The hematologic and clinical chemistry for the blood sample of 8 months before death showed normal creatinine level of 0.4 mg/dL (reference interval: 0.3-1.0 mg/dL) (17). This creatinine value of penguin was in normal range and did not indicate the clinical significance. Since the lack of responsible veterinarian, the patient was treated with systemic antibiotic (enrofloxacin, 15 mg/kg) per oral route for 7 days without other proper treatments. The necropsy was performed in Hanhwa marine Biology Research Center, representative tissues such as liver, kidney, spleen, and stomach were requested to the College of Veterinary Medicine, Jeju National University for histopathologic examination and diagnosis. After the complete necropsy, collected tissue samples were fixed in 10% neutral buffered formalin, trimmed, and embedded in paraffin wax. The paraffin blocks were sectioned at 3 µm, and stained with hematoxylin and eosin (H&E) for light microscopic examination. Special staining such as Congo red stain also performed on paraffin embedded tissue sections of liver, kidney, and spleen. Special stained slides were examined under routine light microscope and by fluorescent microscope under a Texas red filter, as described by Clement and Truong (2).

Grossly, large swelling with central ulceration (moderate bumblefoot = pododermatitis) was existed at the central surface of the right foot (Fig. 1). According to the degree classification by Cooper (3), the foot lesion was classified in grade III. Severe enlarged liver with orange color and blunt edge with firm consistency and marked swelling with pale discoloration of both side kidneys were observed (Fig. 2). Histopathologically, pinkish amorphous materials were observed in perivascular spaces of liver and in the most spaces between the hepatocytes and sinusoids (Fig. 3A). Therefore, many hepatocytes underwent atrophy and degeneration. Marked atrophy of white pulp and diffuse accumulation of pinkish amorphous materials in red pulp and around blood vessels were noted in the spleen (Fig. 3B). Severe multifocal thickening with accumulation of pinkish materials were observed in renal interstitial tissues and around blood vessels (Fig. 3C). Occasionally these amorphous materials also accumulated in some glomerular tufts. The pinkish materials also observed in the lamina propria of stomach (Fig. 3D). The pinkish materials in the liver, kidney, spleen, and stomach were confirmed as amyloid with typical orange color reactions using Congo red stain under the light microscope and with bright red color expressions using Congo red stain under the fluorescence microscope (Fig. 4A-F).

Figure 1. Gross lesions of African penguin. Large swelling with central ulceration at the central surface of the right foot (bumblefoot, grade III).

Figure 2. Gross lesions of African penguin. (A) Severe hepatic swelling with orange color and firm consistency. (B) Enlarge kidneys with pale discoloration.

Figure 3. Histopathologic findings. (A) Liver. Pinkish amorphous materials (arrow) are observed around blood vessels and sinusoids. (B) Spleen. Atrophy of white pulp and diffuse accumulation of pinkish materials (arrow) in red pulp and around blood vessels are noted. (C) Kidney. Pinkish materials (arrow) are observed in renal interstitial tissues and around blood vessels. (D) Stomach. Pinkish materials (arrow) are observed in lamina propria. H&E stain, Scale bars = 50 μm.

Figure 4. Histopathologic findings for the comparison of Congo red-stained amyloid deposits under light microscopy and fluorescence microscopy. Abundant amyloid deposits are clearly seen by light microscopy as an orange color (A, C, E) and fluorescence microscopy as a bright red color (B, D, F). (A, B) Liver. (C, D) Spleen. (E, F) Kidney. Congo-red stain, Scale bars = 50 μm.

Like in mammalian AA amyloidosis, the avian form is mostly systemic with diffuse depositions in many different internal organs except brain (6). Although the limitation of samples in this case, amyloid deposits were nicely observed in most requested tissues such as liver, kidney and spleen. The clinical signs in humans are usually renal symptom with proteinuria due to glomerular amyloid deposits, although other consequences may also develop depending on the tissues of deposition. Clinical signs in systemic amyloidosis of birds seem to be probably difficult to evaluate (6). According to large scaled survey, clinical signs including swelling of the feet, abdominal fullness, dyspnea, and ascites were rarely encountered among the birds with amyloidosis. The presence of amyloidosis was never recognized before necropsy of birds (4).

Based on the survey, 304 (26.5%) out of 1149 birds had amyloidosis (4). The frequency of deposition of amyloid was varied in different internal organs. Amyloid deposition was more frequent in liver (84%), spleen (82%), adrenal gland (71%), and kidney (49%) than other organs such as thyroid (34%), pancreas (31%), intestine (13%), heart (2%) and lungs (0%). The deposits may be either parenchymal (adjacent to sinusoids or capillaries), or vascular (in the inner layer of the media of small arteries and arterioles), or both. Vascular deposition was most prominent in the kidney, where it was often associated with glomerular sclerosis. Parenchymal deposition predominated in the liver, spleen, adrenal, and thyroid. The initially small and scattered deposits in early stage of disease enlarged and eventually coalesced (4). In the present study, the parenchymal deposition of amyloid in liver and spleen was identical with previous literature. However, parenchyma distribution of amyloid was more prevalent than vascular deposition in the kidney of African penguin. Therefore, the deposition of Congo red stain positive materials was more obvious in the renal interstitial tissues and around blood vessels than the glomerular tufts. Renal deposition of amyloid maybe related with the individual difference of bird than the species difference.

Congo red stain is commonly used for the confirmation of amyloid in the tissues and immunohistochemistry using antibody against AA amyloid is an adjunct tool allowing the identification of the type of deposition (1,16). In this study, diffuse amyloid deposition was detected in the liver, kidney and spleen by Congo red staining and fluorescent microscopy. The Congo red-stained amyloid deposits in these organs showed the typical pinkish to pink-reddish color under the light microscopy. When Texas red filter was used with ultra-violet light, the Congo red-stained amyloid showed a bright red color against a dark background.

AA amyloidosis characteristically occurs in humans and animals as a result of a long-standing, chronic infectious or inflammatory process (7). Likewise, avian AA amyloidosis also occurs in birds as a consequence of various inflammatory disorders such as tuberculosis, aspergillosis, bumblefoot, arteriosclerosis and its complications, chronic wound inflammation associated with accidental amputation, and chronic peritonitis caused by a Gram-negative bacillus infection (4,5,8,9,18). Severe pododermatitis (‘bumblefoot’) that progressed to osteomyelitis and then produced amyloidosis in the liver, kidney, spleen, and some intestinal organs in captive zebra finch bird (16). AA amyloidosis is not a rare disease in captive birds and most frequently occurs in waterfowl, which are known to induce AA amyloidosis secondary to ulcerative pododermatitis caused by Staphylococcus spp. infection (8). Secondary amyloidosis associated with inflammatory reaction in pododermatitis also demonstrated in the captive whooper swan in Korea (18). In addition, chicken AA amyloidosis is referred to as amyloid arthropathy in association with Enterococcus faecalis infections (8).

Bumblefoot (pododermatitis) is an inflammatory lesion with or without degenerative changes of avian feet that most commonly arises by the reason of avascular necrosis (13). This disease is a serious problem in several species of penguins (10,14). Based on the previous literatures (3,10,14), the lesion of bumblefoot can be classified in 3 different grades according to its gravity, involved area in foot and prognosis. Grade I include foot pads with mild and localized injury, and grade II had foot pads with more extensive injury and bacterial infection. An extension of the lesions and presence of an infectious and degenerative process with high temperature and pain are observed in the foot with grade III. Although unproven, captive penguins are prone to the lesions of bumblefoot due to the increased sedentary habits, changes in normal activity patterns in living, less time swimming in the water, and prolonged time in hard and abrasive surfaces or surfaces with excessive moisture or fecal contamination (14). This disease is characterized by excoriations, ulcerations and plantar epithelial abscess in ventral surface of feet. If untreated or inadequately treated, pododermatitis can lead severe complications such as deep-seated abscess or granulomas, osteomyelitis, and eventually death. Penguins with bumblefoot can show the clinical signs of abnormal stance and gait, increased lying down, and footpad ulceration.

According to previous literatures, several approaches to the treatment of bumblefoot in wild birds were demonstrated (13,15). A 4-pronged therapeutic regime composed of 1) systemic broad spectrum antibiotic therapy, 2) direct intralesional long-term antibiotic delivery, 3) surgical debridement, and 4) postoperative protective foot shoe or casting offered the most effective result for the majority of bumblefoot cases of raptors (15). Clinicians in SeaWorld California developed a treatment protocol for bumblefoot in penguins that involves surgical debridement, flushing, complete closure, topical wound dressings, and bandage changes every 2 to 3 days. Due to bumblefoot lesions heal very slowly, long-term protection plan of the foot is necessary. Hence, at 10 to 14 days after surgery, the penguin is fitted with a neoprene bootie, or “iceflo”, which can be worn for 2 to 3 months to protect the surgical site and improve healing (13).

To reduce the prevalence of bumblefoot lesions in captive penguins in zoologic parks and aquarium, good preventive measures are necessary. The longer penguins stay on land and the substrate quality is poor, the more the lesions of bumblefoot may occur. Therefore, aquatic environmental enrichment allowed captive penguins to spend more time in the water, favoring the reduction of the bumblefoot lesions and even healed the wounds of penguins (13,14). In addition, continuous monitoring for the foot lesions in bird will be required, especially in captive birds of zoo and aquarium.

This research was supported by the 2022 scientific promotion program funded by Jeju National University.

The authors have no conflicting interests.