Conference 05 - 2013 Case: 02 20131016

Signalment:

Wild-caught juvenile male raccoon, (Procyon lotor)A juvenile male raccoon exhibiting neurological deficits was found by a member of the public and submitted to a wildlife rehabilitation facility in northern California in January 2012. The raccoon had wounds on the tail, pale mucous membranes, ataxia, head tremors, mild inappetence and an initial exacerbated startle response. The animal could never right himself, rolled over and was uncoordinated. Palliative treatment with meloxicam (Metacam-«, 0.2 mg/kg subcutaneously q24h), procaine G penicillin (20,000 Units/kg subcutaneously q24h), iron dextran (10 mg/kg intramuscularly once) and vitamin B complex (30 mg/kg subcutaneously once), did not ameliorate the clinical signs. The raccoon was humanely destroyed and submitted to the California Animal Health and Food Safety Laboratory, Davis, California, for necropsy examination.

Gross Description:

The raccoon had adequate fat stores. The liver was diffusely and markedly enlarged and pale with irregular, undulating surfaces. The spleen was also markedly enlarged and meaty. All lymph nodes noted were very pale and enlarged. The lungs were collapsed and there were occasional pinpoint pale subpleural foci. The gastrointestinal tract contained scant contents with a small amount of dry fecal matter in the distal large intestine.

Histopathologic Description:

In the section of cerebellum the neuronal as well as glial cell cytoplasm was markedly distended (up to 3 times normal size) by aggregates of delicate clear round vacuoles (approximately 1μm in diameter). These aggregates occasionally displaced the nucleus to the periphery of the cell. Multifocally swollen eosinophilic axons were observed in the granular layer. In the section of the spleen, foamy macrophages expanded the germinal centers and formed extensive sheets that replaced and effaced the red pulp.

Morphologic Diagnosis:

Cerebellum: Severe diffuse neuronal and glial cell vacuolation and swelling with occasional multifocal spheroids (suspect storage disease).
Spleen: Germinal centers and red pulp severe diffuse histiocytosis (suspect storage disease).

Lab Results:

Test	Result
Aerobic culture- lung, liver, mesenteric lymph node	Mixed growth
Fecal PCR for Salmonella sp.	Salmonella arizonae
Fecal flotation	Negative
Serology for Toxoplasma sp.	Negative
Heavy/trace mineral analysis (lead, manganese, iron, zinc, arsenic, cadmium, molybdenum, copper, mercury, selenium)	Within normal limits
Rabies testing via fluorescent antibody testing on brain tissue	Negative
Lysosomal enzyme analysis	See Table 1
Special stains: Oil red O (on formalin fixed frozen sections), PAS, Sudan Black, Luxol fast blue and acid fast	- Multifocal endothelial cell intra-cytoplasmic accumulation of Oil red O-positive material - Affected neurons and macrophages were Oil red O, PAS, Sudan Black, Luxol fast blue and acid fast negative
Autofluorescence (via UV scope)	Negative

Condition:

Nieman-Pick Disease; Sphingomyelin lipidosis

Contributor Comment:

In addition to the cerebellum and spleen, multiple tissues were infiltrated and expanded by aggregates or sheets of previously described foamy macrophages. These include multiple lymph nodes; lamina propria of the tongue, intestine, colon; portal areas of the liver; and around the pulmonary vessels, in the alveolar spaces and subpleurally in the lung. Additionally, cerebral neurons and glial cells as well and peripheral ganglia neurons were similarly affected. Various degrees of cytoplasmic foaminess were also observed in the epithelial cells of the renal tubules, parietal glomerular cells and hepatocytes.

Transmission electron microscopy revealed lysosomal accumulations of floccular variably electron dense and frequently concentrically arranged lamellar material consistent with lysosomal storage disease. However, ultrastructural analysis is relatively non-specific regarding type of storage.

Measurement of lysosomal enzyme activity including sphingomyelinase, β-galactosidase, β-hexosaminidase and β-hexosaminidase A and B was performed in water homogenates of the brain samples from affected and age-matched non-affected raccoon(8) (Table 1). This assay revealed complete absence of sphingomyelinase activity. The absence of sphingomyelinase activity is a criterion for the diagnosis of sphingomyelin lipidosis (also known as Niemann Pick disease (NPD)).(7)

Sphingomyelin lipidosis belongs to a large group of sphingolipidosis lysosomal storage diseases that also includes GM1 and GM2 gangliosidosis and globoid cell leukodystrophy, to name a few. In sphingolipidoses the spectrum of affected organs is wide and often includes viscera and macrophages because the substrate is derived from all cell membranes.(3) The involvement of neurons in most LSDs is due to both the high metabolic activity of these cells and their long life span, which allows the gradual accumulation of undegraded substrate.(3) Ultrastructural pathology offers useful information in the diagnosis of LSDs and helps categorize the type of LSD. In diseases accumulating sphingolipids, storage bodies are characterized by membranous material arranged concentrically (membranous cytoplasmic bodies). None of these forms is specific for a given disease, but concentric lamellae are most common in GM1 and GM2 gangliosidoses. In the present case the storage bodies were poorly defined, concentrically arranged lamellar whorls. Histochemistry, immunohistochemistry and fluorescence microscopy may also be of use in identifying storage material, but definitive and gold standard for diagnosis is by means of biochemical analysis.(3)

There is no single presentation common to all lysosomal storage diseases; the clinical and gross pathological manifestations are dependent on the deficient enzyme and the outcome of the deficiency in the organs that utilize the enzyme. Microscopically, LSDs are characterized by accumulation of enlarged lysosomes containing uncatabolized substrate in solution or complexed with related chemical species, which will be partially or wholly removed during fixation and preparation of the paraffin wax-embedded sections. If the substrate is soluble in water or lipid solvents, there will be a vacuolated appearance of the affected cells.(3)

Sphingolipids are an important group of structural lipids in which the unifying compound, ceramide, is esterified to sialyloligosaccharides to form gangliosides, to other saccharides to form neutral glycolipids such as globoside, or to phosphocholine to form sphingomyelin.(3)

The primary metabolic defect in NiemannPick disease (NPD) types A and B in man is the lack of sphingomyelinase enzyme that catalyzes the hydrolytic cleavage of sphingomyelin to ceramide and phosphocholine.(3) The reduced or absent enzyme activity results in accumulation of sphingomyelin in lysosomes.(7) Similarly to humans, an autosomal recessive mode of inheritance has been demonstrated in a cat and a dog.(2,8)

Similar histological and ultrastructural features to those in this case were reported affecting neurons, oligodendroglial cells, macrophages, renal epithelial cells, endothelium and pericytes in the CNS, PNS, spleen, liver, lung and kidney in a cat,(1) a dog and a Hereford calf.(6) Consistent with the present case, these animals presented as juveniles, had neurological signs and virtually no sphingomyelinase activity was detected in the brain and liver compared with normal controls. Autofluorescence by UV light was reported in a dog with NPD,(2) but was not found in the present case.

Table 1: Lysosomal enzyme activities in brain homogenates from the affected and a normal raccoon

Enzyme activity (nmol/h/mg protein)	Affected Raccoon	Normal Raccoon
Beta-galactosidase^a	64.0	22.6
Beta-hexosaminidase A^b	81.4	28.9
Beta-hexosaminidase A and B^c	611.1	118.0
Sphingomyelinase^d	0.13 (3X)	5.2

a- measured using 4MU-β-galactoside
b- measured using 4MU-β-N-acetylglucosaminideSO4
c- measured using 4MU-β-N-acetylglucosaminide
d- measured using 14C-sphingomyelin; 3x means measured three times

JPC Diagnosis:

Cerebellum: Neuronal, glial cell and endothelial cytoplasmic vacuolation, diffuse, marked, with gliosis.
Spleen: Histiocytosis, diffuse, marked with cytoplasmic vacuolation.

Conference Comment:

Conference participants briefly reviewed select inherited (see Table 2) and acquired lysosomal storage diseases of veterinary importance. Sphingolipidoses result from defective catabolism of normal cell membrane constituents known as glycosphingolipids, and exhibit autosomal recessive inheritance. This case is an excellent example of sphingomyelinosis, or Niemann-Pick disease, the pathogenesis of which is thoroughly described in the contributors comment. GM₁ gangliosidosis has been described in dogs, cats, Friesian cattle and sheep. Accumulation of lysosomal GM₁ ganglioside occurs due to a deficiency in β-galactosidase, though GM₁ gangliosidosis in Suffolk sheep is actually due to deficiencies in both β1-galactosidase and α-neuraminidase. GM₂ gangliosidosis results from insufficient activity of hexosaminidase (which exists as an αβ- or ββ-dimer) or its activator protein. This condition is reported in domestic shorthair and Korat cats, German shorthaired pointers and golden retrievers due to a β-subunit deficiency, while in Japanese spaniel dogs and Yorkshire pigs there is an activator protein deficiency. Tay-Sachs and Sandhoff diseases are examples of GM2 gangliosidosis in humans. As in sphingomyelinosis, neurons in GM₁/GM₂ gangliosidosis are expanded by abundant, foamy, PAS-positive cytoplasm, with faint granules; ultrastructurally lysosomal granules are composed of concentric membranous whorls. Glial cells and macrophages are also affected. Glucocerebrosidosis, which is similar to Gaucher disease in humans, has been reported in Sydney Silky Terriers, and results from deficient glucocerebrosidase, the catalyst for conversion of glucocerebroside to ceramide. Microscopically, glucocerebrosidosis manifests in hepatic and lymph node sinusoidal macrophages, as well as some neurons, but not in Purkinje cells or the spinal cord. Ultrastructurally, storage material appears twisted or branching.(5)

Globoid cell leukodystrophy, also known as galactocerebrosidosis, is an autosomal recessive disorder reported in dogs, cats and polled Dorset sheep. It is classified within the sphingolipidosis group of storage diseases and results from deficient activity lysosomal galactocerebrosidase, an enzyme which normally catalyzes the breakdown of galactocerebrosides. Galactocerebrosides are important components of myelin, but at high concentrations are cytotoxic; excessive accumulation in oligodendrocytes and Schwann cells causes extensive cellular degeneration and necrosis, halting active myelination. This combined with the degeneration of existing myelin, results in demyelination and axonal loss. Phagocytic macrophages, however, are unable to degrade galactocerebroside, and thus appear microscopically as characteristic swollen, PAS-positive globoid cells, which exhibit perivascular cuffing within the white matter. In contrast to many of the other lysosomal storage diseases, neurons are not typically involved in the accumulation of excess storage material in galactocerebrosidosis.(5)

Glycoproteinoses, such as α- and β-mannosidosis, or α-L-fucosidosis, are characterized by defective degradation of the carbohydrate component of N-linked glycoproteins. In α-mannosidosis, a historically important entity in Angus cattle, a defective enzyme leads to decreased lysosomal α-mannosidase activity in all cells except hepatocytes, which leads to widespread mannose/N-acetylglucosamine oligosaccharide deposition. β-mannosidosis due to β-mannosidase deficiency is reported in Salers cattle and Nubian goats. In both α- and β-mannosidoses, neurons, macrophages and secretory epithelial cells are most severely affected, although storage material is typically lost during tissue processing so vacuoles appear empty on standard H&E slides. In α-L-fucosidosis deficient activity of α-L-fucosidase leads to a similar histological appearance; this condition is autosomal recessive in English springer spaniels.(5)

Mucopolysaccharidoses are distinguished by defective catabolism of glycosaminoglycans, so skeletal and connective tissue abnormalities such as deformities, degenerative joint disease, and thickening of the heart valves or leptomeninges are often observed; neurons can be involved as well. Deficiency in α-L-iduronidase, known as mucopolysaccharidosis type I (MPS I) in humans, is reported in domestic shorthair cats and Plott hounds. Storage primarily occurs in mesoderm-derived cells. Deficiency in N-acetylglucosamine-6-sulfatase leads to the veterinary counterpart of human MPS III, which has been described in Nubian goats. Mesoderm-derived cells are packed with heparan sulfate, while neurons contain gangliosides, which accumulate due to interference with neuraminidase activity. Siamese and domestic shorthair cats occasionally have a deficiency in arylsulfatase-B, a disorder known as MPS VI in humans. Mucopolysaccharidosis VI differs from MPS I and II in that neuronal storage does not occur. Finally, the counterpart to human MPS VII is reported in dogs and cats secondary to a lack of β-glucuronidase. Again, microscopic findings are similar to those described above; however, there is also widespread neurovisceral storage.(5)

Glycogenoses result from defective glycogen catabolism. α-1,4-glucosidase deficiency, an autosomal recessive condition documented in shorthorn and Brahman beef cattle, leads to widespread glycogen storage, both within lysosomes and intracytoplasmically. In contrast, other types of glycogen storage diseases are concentrated primarily within the liver and muscle. Since the excess storage material is composed of glycogen, it is PAS-positive and diastase-sensitive. Neurons are severely affected in this disease.(5)

Acquired lysosomal storage diseases often follow the ingestion of toxic plants, or, less commonly, drug administration. Swainsonine is an indolizidine alkaloid found in several plant species, such as locoweed (Astragalus, Oxytropis sp.). Ingestion of swainsonine by grazing livestock causes inhibition of α-mannosidase, inducing a form of α-mannosidosis. Ingestion by pregnant sheep can also result in abortion and fetal malformation. Ingestion of Trachyandra divaricata or T. laxa causes excessive storage of lipofuscin in central and peripheral neurons of South African/Australian livestock.(5) Aminoglycosides, such as gentamicin, accumulate within lysosomes of renal proximal tubular cells where they inhibit lysosomal phospholipases, producing aggregates of phospholipid-containing myeloid bodies. As lysosomes become progressively distended with myeloid bodies, they rupture, releasing acid hydrolases as well as high concentrations of aminoglycosides into the cytoplasm, further damaging the cell.(4)

Regardless of the underlying genetic or acquired cause, most lysosomal storage diseases in veterinary species are characterized clinically by an early onset of neurologic impairment. Considering the similarity of clinical signs as well as the frequent overlap of the gross and microscopic lesions in many types of lysosomal storage diseases, electron microscopy and especially measurement of lysosomal enzyme activity are often necessary to elucidate a specific etiology.

Table 2: Select inherited lysosomal storage diseases(5)

Condition	Enzyme Defect	Storage Material	Inheritance/species
GM1 gangliosidosis	β-galactosidase	GM₁ ganglioside in lysosomes of neurons, glial cells, macrophages	- autosomal recessive - dogs, cats, Friesian cattle; - suffolk sheep - deficiencies in β1-galactosidase AND α-neuraminidase.
GM₂ gangliosidosis (Tay-Sachs and Sandhoff diseases)	-hexosaminidase (αβ- or ββ-dimer) -activator protein	GM₂ ganglioside in lysosomes of neurons, glial cells, macrophages	- autosomal recessive 1. domestic and Korat cats, German shorthaired pointers, golden retrievers: β-subunit deficiency 2. Japanese spaniel dogs, Yorkshire pigs: activator protein deficiency
Sphingomyelinosis (Niemann-Pick disease)	sphingomyelinase	sphingomyelin in lysosomes of neurons and macrophages	- autosomal recessive in cat and dog
Globoid cell leukodystrophy (glactocerebrosidosis)	galactocerebrosidase	galactocerebrosides in oligodendrocytes/Schwann cells, globoid cell macrophages (NOT in neurons) demyelination, axonal loss	- autosomal recessive - dogs, cats and polled Dorset sheep
Glucocerebrosidosis (Gaucher disease)	glucocerebrosidase	glucocerebroside in lysosomes of hepatic/lymph node sinusoidal macrophages, some neurons (NOT in Purkinje cells or the spinal cord)	- Sydney Silky Terriers
α-Mannosidosis	α-mannosidase	mannose/N-acetylglucosamine oligosaccharide in lysosomes of neurons, macrophages, secretory epithelial cells	- Angus cattle
β-Mannosidosis	β-mannosidase	oligosaccharides in lysosomes of neurons, macrophages, secretory epithelial cells	- Salers cattle and Nubian goats
α-L-fucosidosis	α-L-fucosidase	- fucose containing glycoconjugates in lysosomes of neurons - similar appearance to α/β-Mannosidosis	- autosomal recessive - English springer spaniels
MPS I	α-L-iduronidase	mucopolysaccharide storage in mesoderm derived cells	- domestic shorthair cats and Plott hounds
MPS III	N-acetylglucosamine-6-sulfatase	heparan sulfate in mesoderm-derived cells; neurons contain gangliosides	- Nubian goats
MPS VI	arylsulfatase-B	mucopolysaccharide storage in mesoderm derived cells; neuronal storage does not occur	- Siamese and domestic shorthair cats
MPS VII	β-glucuronidase	widespread neurovisceral storage	- dogs and cats
Glycogenosis (type II in humans)	α-1,4-glucosidase	widespread glycogen storage within lysosomes and intracytoplasmically; including neurons	- autosomal recessive in shorthorn and Brahman beef cattle

References:

1. Baker HJ, Wood PA, Wenger DA, Walkley SU, Inui K, Kudoh T, et al. Sphingomyelin lipidosis in a cat. Vet Pathol. 1987;24:386-391.

2. Bundza A, Lowden JA, Charlton KM. Niemann-Pick disease in a poodle dog. Vet Pathol. 1979;16:530-538.

3. Jolly RD, Walkley SU. Lysosomal storage diseases of animals: an essay in comparative pathology. Vet Pathol. 1997;34:527-548.

4. Kaloyanides GJ. Drug-phospholipid interactions: role in aminoglycoside nephrotoxicity. Ren Fail. 1992;14(3):351-357.

5. Maxie MG, Youssef S. Nervous system. In: Maxie MG, ed. Jubb, Kennedy and Palmers Pathology of Domestic Animals. 5th ed. Vol 1. St. Louis, MO: Elsevier; 2007:322-332, 381.

6. Saunders GK, Wenger DA. Sphingomyelinase deficiency (Niemann-Pick disease) in a Hereford calf. Vet Pathol. 2008;45:201-202.

7. Stanbury JB, Wyngaarden JB, Fredrickson DS. The Metabolic Basis of Inherited Disease. 3rd ed. New York, NY: McGraw-Hill; 1972.

8. Wenger DA, Sattler M, Kudoh T, Snyder SP, Kingston RS. Niemann-Pick disease: a genetic model in Siamese cats. Science. 1980;208:1471-1473.