Lysosomal storage diseases have been reported in humans, domestic animals, and emus. Most of these diseases have an autosomal recessive mode of inheritance and cause morbidity and mortality in juveniles. At the Smithsonian National Zoological Park (SNZP), two American flamingos (Phoenicopterus ruber) were presented for necropsy after a 1- to 14-week history of inappetence, self-isolation, head tremors, and an inability to rise. Gross necropsy findings were not dramatic, but histology revealed extensive, foamy vacuolation of Purkinje cells of the cerebellum, and neurons and glia of the cerebrum, and peripheral ganglia. Neurons and glia of the affected brains did not stain with PAS or toluidine blue, but had positive intracytoplasmic staining with Luxol fast blue. These results, concurrent with the identification of whirled, lamellar, and membrane bound structures within the cytoplasm of affected neurons and astroglia by transmission electron microscopy, supported gangliosidosis as a probable causative entity. Further evaluation of tissues currently being undertaken will more precisely determine the enzyme deficiency. It is likely that these two birds were related.
Lysosomal storage diseases (LSDs) have been reported in humans, domestic animals, and emus.1,2,4 The most common LSDs are the gangliosidoses and the mannosidoses. These diseases generally have an autosomal recessive mode of inheritance.5,6 However, animals can acquire one type of mannosidosis by ingesting certain toxic plants.5,6 Gangliosidoses have been reported in humans, dogs, cats, cattle, sheep, pigs, and emus, and are caused by a deficiency of beta-galactosidase (GM1-gangliosidosis) or hexosaminidase (GM2-gangliosidosis).1,2,5 Accumulation of lysosomes containing storage material primarily occurs within the neural tissue; this appears histologically as foamy expansion of the cytoplasm of neurons and glial cells. Clinical signs include head tremors, ataxia, and behavioral changes. Alpha and beta mannosidosis are caused by a defect in alpha or beta mannosidase, respectively.5 These have been recognized in humans, cats, cattle, and goats. Affected individuals show clinical signs similar to those of the gangliosidoses, but storage of mannose compounds occurs in macrophages, fibroblasts and epithelial cells, as well as within neurons.
The following report outlines a lysosomal storage disease in two American flamingos (Phoenicopterus ruber) at the Smithsonian National Zoological Park (SNZP) in Washington, DC.
In July of 2000, a 6-week-old, female, American flamingo was reported by keepers to be inappetent, lethargic, and aloof from the rest of the flock. The bird appeared smaller than other juveniles of similar age and was considered slow to wean from hand rearing. The animal was treated for 5 days with enrofloxacin PO and seemed to improve. A polymerase chain reaction (PCR) test for flamingo alpha herpesvirus was negative. Two months later, the bird was again reported to display aloofness, decreased food consumption, and increased time sleeping. Clinical signs were progressive over the following 3 weeks despite treatment with enrofloxacin, stanozolol, fluconazole, acyclovir, clindamycin, ceftiofur, carafate, nystatin, and vitamins E and B. A PCR test for flamingo alpha herpesvirus was again negative and there were no consistent or progressive hematologic abnormalities during the illness. In the 2 weeks prior to death, the bird showed abnormal head positioning and movement, appeared ataxic, and had difficulty rising. Three and a half months after the initial presentation the animal was found down and unable to maintain a standing position when lifted. Euthanasia was elected.
Necropsy revealed an animal in good nutritional condition with moderate hepatomegaly and cerebral dural vessel injection and hemorrhage. The most striking histologic finding was diffuse, foamy, cytoplasmic vacuolation of the cerebellar Purkinje cells and cerebral neurons and astrocytes. Peripheral nerve ganglia and plexuses had similar changes. Multifocally, Purkinje cells were reduced in number and had decreased arborization. The material within the cytoplasm of neurons and astrocytes was not stained by PAS or toluidine blue, but was stained by Luxol fast blue. Neurons and glia of age-matched, normal American flamingos were negative with all three special stains.
A section of paraffin embedded cerebral tissue was removed from the block and processed routinely for transmission electron microscopy (EM). By EM, the cytoplasmic vacuoles consisted of two distinct structures that were only occasionally present within the same cell. In greater abundance were clear, membrane-bound vacuoles. Vacuolar membranes were occasionally ruptured or reduplicated. Fewer cells contained whirled or lamellar structures adjacent to the nucleus.
In November of 2001, a 5-month-old, male, American flamingo was reported to be isolating itself from the group and showing decreased grooming activity. This bird had been given a 10-day treatment of trimethoprim-sulfadiazine 3 weeks previously for an undisclosed reason. It had been previously housed with two other juvenile flamingos that died due to pneumonic aspergillosis. A blood sample revealed slight hypoglycemia (127 mg/dl; normal 134–276 mg/dl) and a mild increase in hematocrit (49.0%; normal 29.0–48.5%), but was otherwise normal. Two days after presentation the bird was found down, unable to stand and too weak to eat. Aspergillosis was suspected and the animal was euthanatized.
Necropsy revealed abundant fat stores and pale streaks on the cardiac and skeletal muscles. Histologically, these muscle changes were consistent with mild capture myopathy. Cerebellar and cerebral changes were similar to that in case 1, as were the staining characteristics of the stored material. EM was not performed on this animal, but virus isolation and PCR for West Nile virus were performed on frozen tissues and were negative.
Clinical signs and histologic lesions in these two flamingos were consistent with a LSD. Electron microscopic examination of samples from one bird revealed whirled, lamellar and membrane-bound, clear vacuoles within neurons and astroglia; these findings most closely resemble the lysosomal storage vacuoles present in gangliosidosis.3 Positive histochemical staining of the stored material with Luxol fast blue is also indicative of a defect in lipid degradation.4
Gangliosidoses in other species are usually associated with an autosomal recessive, inherited enzyme defect. American flamingos at the SNZP are housed in a flock of approximately 50 birds, with natural breeding. Parentage of the affected flamingos is unknown. It is possible that these two birds, which were born 1 year apart, were full siblings.
The population of American flamingos at the SNZP is relatively stable and this disease had not been seen before the first case presented to the pathology department. The last birds that were added to the group arrived in September of 1996 and consisted of two males, and three females. All of these animals are still alive and potentially in the breeding pool. It is possible that one or more of these new arrivals possess the mutated gene. Alternatively, a random mutation may have occurred in the existing flock and homozygosity may have occurred through inbreeding.
Definitive identification of the defective enzyme requires biochemical evaluation of unfixed nervous tissue. These evaluations are currently being undertaken and should shed light on this newly recognized lysosomal storage entity in American flamingos.
The authors appreciate the assistance of Mr. John Jenkins in expertly preparing the electron micrographs, and Histoserv, Inc. of Gaithersburg, MD, for histologic processing. This work was supported by a grant from Friends of the National Zoo.
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