A pathologic condition called “avian amyloidosis” develops in domestic ducks as a result of persistent inflammation and infectious or cancerous situations¹. The condition is characterized by the extracellular deposition of an insoluble substance called amyloid within various tissues and organs of the body¹. Amyloid is an eosinophilic homogenous material derived from the protein precursors with a characteristic fibrillar pattern². The most common type of amyloid in birds is found to be AA-type, derived from an acute-phase reactant serum amyloid A (SAA)³. Although the pathogenesis of avian amyloidosis was not clearly explored, some predisposing factors, such as chronic inflammation and infections, could lead to the accumulation of amyloid in various tissues³. Diagnosis of amyloidosis occurs rarely in antemortem due to nonspecific clinical signs. Hence, histopathological studies are required for its confirmation. The present article describes the gross and histopathological characteristics of amyloidosis with fibrosis secondary to ulcerative pododermatitis in a Vigova duck.

A 2-year-old Vigova duck that had been raised under an intensive system and weighed 3.2 kg was brought in for a postmortem examination to the Department of Veterinary Pathology at the College of Veterinary and Animal Sciences in Pookode. The duck had a history of lameness, lethargy, decreased egg production, foot swelling, and an enlarged abdomen. External examination of the carcass revealed an enlarged abdomen with ventral lacerations and swelling on the plantar region of the feet, thickening of the skin, and a centrally swollen necrotizing crater-forming ulcer. At necropsy, a severely enlarged, pale pink, firm liver occupied almost the entire length of the abdominal cavity. The surface of the liver was pale pink, smooth, and glistening, with focal capsular thickening and rounded edges. The liver was 440 g in weight and its caudal end was well past the sternum (Fig. 1A). Moderate splenomegaly, diffuse gastrointestinal serosal congestion, and engorged mesenteric blood vessels were among the other findings. There were hemorrhagic mucoid materials inside the intestinal lumen. The spleen and kidney were enlarged and congested. The ovary was moderate to severely atrophied and hyperaemic (Fig. 1B). In the leg (Fig. 1C), the plantar swelling's opening revealed that there was gooey pus present in the cavity (Fig. 1D). For histopathological analysis, tissue samples were gathered and preserved in 10% neutral buffered formalin. The tissues that had been treated with formalin were prepared for paraffin embedding. Hematoxylin and eosin were used to stain sections that were 5 pm thick. Congo red and Masson's trichrome were used to further stain serial tissue sections. Tissue from plantar foot were stained with Brown and Brenn method⁴.

Microscopically, in the liver, moderate to severe deposition of eosinophilic amorphous substances in the 'space of Disse', periportal, and perivascular areas with effacement of hepatocytes was noticed. Large amounts of amorphous pink deposits obliterated the sinusoidal spaces, leading to compression atrophy and degenerative changes in hepatocytes (Fig. 2A). Islands of atrophied hepatocytes with diffuse infiltration of mononuclear cells, such as plasma cells, and lymphocytes with fewer heterophils were present in the hepatic parenchyma. The amorphous deposition of liver turned orange to red when stained with Congo red, confirming that it was amyloid deposition⁴.

Amyloid accumulation in the spleen led to hyalinization of the walls of ellipsoids and penicillar capillaries. (Fig. 2B). Also, multifocal nodular deposition was noticed in the red pulp region of the spleen, along with disruption and effacement of the ellipsoidal and periellipsoidal white pulp by amorphous eosinophilic substances. Histopathologically, the ovary showed congestion and thickening of the blood vessel wall in addition to atrophied ovarian follicles. A moderate amount of amyloid deposition was noticed focally in the interstitium and around the wall of the thickened blood vessels (Fig. 2F). In kidneys, a mild amount of amyloid deposition was found around the walls of the renal blood vessels and tubular basement membrane. Desquamation of tubular epithelium and degenerative changes of renal tubules with infiltration of mononuclear cells were evident. Diffuse, moderate amyloid deposits were present in the lamina propria of the duodenum with severely congested blood vessels and infiltration of mononuclear cells such as plasma cells and lymphocytes. Further staining with Masson's trichrome revealed that the amyloid-free collagen fibre stained blue while the amyloid-bound collagen fibre stained red in other organs (Fig. 3A-C). In addition to amyloid deposition, the presence of collagen deposits confirmed fibrosis along with amyloidosis in the present case⁵. Further staining with Congo red and Masson's trichrome on tissue sections of other organs also revealed amyloid deposition with fibrosis.

Bacteriological culture of pus drained from plantar abscess produced round, smooth glistening white colonies of 1-4 mm size on nutrient agar (Fig. 4A). On Gram staining of smear from culture revealed group of cocci arranged in bunches indicating Staphylococcus species. Histopathologic examination of plantar swelling revealed mild diffuse fibrosis of the dermis around the necrotic area with heterophil infiltration and cocci bacteria (Fig. 4B). Gram-positive cocci were stained blue with the Brown and Brenn method of staining (Fig. 4C).

Although there are different biochemical types of amyloids exists in animals, the reactive type or AA amyloid type protein deposits predominate, which was made up of acute phase protein, serum amyloid A (SAA). Only systemic AA amyloidosis in domestic and caged wild birds has been documented⁵. Our observation of amyloid deposition in systemic organs other than the liver secondary to the chronic inflammatory bumble foot condition was in agreement with the observations of earlier reports⁶, ⁷. The precursor protein SAA required for amyloid deposition in various tissues and organs were upregulated due to chronic inflammatory stimuli produced by pododermatitis condition on foot. Liver abnormalities, both gross and microscopic, were consistent with prior reports of severe hepatic amyloidosis in ducks⁸, ⁹. Systemic amyloid deposition in liver and other organs such as ovary, kidney, spleen, pancreas and adrenals were reported in Japanese quail and Bengalese finch¹⁰, ¹¹. Avian amyloidosis is systemic type in nature and its development is associated with predisposing conditions such as age, breed, neoplastic conditions, stress conditions, chronic inflammation and infections⁸. A study on management of bumblefoot in duck demonstrated that Staphylococcus is one of the infectious agents in the digital pad leading to chronic inflammation of the foot¹¹. Since amyloid has an affinity to collagen¹², it is essential to assess the quantity of amyloid bound with collagen fibers to find the severity of damage caused by amyloid deposition in the connective tissue stroma of various organs. When stained with trichrome, amyloid that was attached to collagen fibers appeared red while amyloid without collagen fibre appeared blue¹³. Although the gold standard for amyloid detection remains Congo red staining, trichrome staining is an effective method to differentiate amyloid bound with or without collagen fibers¹³. Therefore, it is more appropriate to perform both trichrome and Congo red staining in amyloidosis to quantify the percentage of fibrosis in organs. Avian amyloidosis is a progressive fatal disease condition and amyloid fibrils deposited in tissues are relatively insoluble and resistant to physiological breakdown¹⁴. Due to the possibility of oral and interspecies amyloid transfer¹⁵, it is required to conduct additional research on amyloidosis in birds used for food. Moreover, it is critical to distinguish between amyloid deposits, fibrosis, and their combination or extent in order to completely comprehend systemic amyloidosis in ducks.