Disseminated blastomycosis in two California sea lions (Zalophus californianus).

Two captive California sea lions (Zalophus californianus) from different facilities were diagnosed with disseminated blastomycosis. The first, a 12-yr-old male, died after a 3-wk history of progressive anorexia and lethargy. Gross examination revealed acute jejunitis with focal perforation and associated peritonitis, along with severe purulent bronchopneumonia. The second, a 15-yr-old female, was euthanized after a 2-wk history of severe cutaneous ulceration and declining clinical condition. Gross examination revealed severe pyogranulomatous bronchopneumonia and ulcerative dermatitis. Histopathologic examination in both individuals revealed severe multifocal subacute to chronic pyogranulomatous pneumonia associated with massive numbers of fungal organisms morphologically compatible with Blastomyces sp. Fungal organisms were 8-20-microm-diameter broad-based budding yeasts with thick, refractile, double-contoured walls. The male sea lion had multifocal transmural Blastomyces-induced enteritis with subsequent rupture and peritonitis. The organism was also present in the liver, with minimal associated inflammation. The female had severe multifocal pyogranulomatous ulcerative dermatitis associated with large numbers of intralesional fungal organisms. Dissemination to the spleen had occurred in both animals. A serologic immunodiffusion test for Blastomyces dermatitidis was positive in the male. The presumptive primary pathogen in both cases was Blastomyces dermatitidis.

Gastric spiral bacteria in small felids.

Nine small cats, including one bobcat (Felis rufus), one Pallas cat (F. manul), one Canada lynx (F. lynx canadensis), two fishing cats (F. viverrina), two margays (F. wiedii), and two sand cats (F. margarita), necropsied between June 1995 and March 1997 had large numbers of gastric spiral bacteria, whereas five large cats, including one African lion (Panthera leo), two snow leopards (P. uncia), one Siberian tiger (P. tigris altaica), and one jaguar (P. onca), necropsied during the same period had none. All of the spiral organisms from the nine small cats were histologically and ultrastructurally similar. Histologically, the spiral bacteria were 5-14 microm long with five to nine coils per organism and were located both extracellularly within gastric glands and surface mucus, and intracellularly in parietal cells. Spiral bacteria in gastric mucosal scrapings from the Canada lynx, one fishing cat, and the two sand cats were gram negative and had corkscrewlike to tumbling motility when viewed with phase contrast microscopy. The bacteria were 0.5-0.7 microm wide, with a periodicity of 0.65-1.1 microm in all cats. Bipolar sheathed flagella were occasionally observed, and no periplasmic fibrils were seen. The bacteria were extracellular in parietal cell canaliculi and intracellular within parietal cells. Culture of mucosal scrapings from the Canada lynx and sand cats was unsuccessful. Based on morphology, motility, and cellular tropism, the bacteria were probably Helicobacter-like organisms. Although the two margays had moderate lymphoplasmacytic gastritis, the other cats lacked or had only mild gastric lymphoid infiltrates, suggesting that these organisms are either commensals or opportunistic pathogens.

Gastric spiral bacteria and intramuscular sarcocysts in African lions from Namibia.

Six juvenile male, one adult male, and three adult, female African lions (Panthera leo) from Etosha National Park, Republic of Namibia were presented for necropsy. Two of four adults and one of six juveniles had moderate numbers of gastric spiral bacteria. Additionally, four of four adults had sarcocysts. All juveniles had enteric Sarcocystis sp. oocysts, but no sarcocysts. The gastric spiral bacteria were located extracellularly in fundic and pyloric glands, and also apparently intracellularly within parietal cells in the fundic region. The organisms were 4 to 8 microns long, 0.63 micron wide, with a periodicity of 0.60 micron. The bacteria had blunted ends with multiple flagella. No periplasmic fibrils were observed. The histologic and ultrastructural characteristics of the bacteria were considered most consistent with species in the genus Helicobacter or incompletely identified Helicobacter-like organisms. Gastric inflammation did not differ significantly between infected and uninfected individuals. The bacteria may be commensals, or an opportunistic pathogen. The sarcocysts were observed in hindlimb skeletal muscle of four individuals, with one individual also containing a single sarcocyst within glossal musculature. All observed cysts were mature, and were contained within individual myocytes. The cyst wall consisted of a 44 to 66 nm, granular, electron dense parasitophorous membrane with subjacent, 0.8 to 1.3 microns thick, granular and fibrillar ground substance which also extended into the cyst interior as thin septa. The membrane was folded and lined irregularly spaced, 0.8 to 1.3 microns tall villi centrally containing ground substance. The membrane was continuous in the villar projections, but divided into discrete aggregations of the electron dense material between the villi. Bradyzoites within the interior of the cyst were 3 by 12 microns. The sarcocysts were determined to be Sarcocystis felis based on the characteristic ultrastructural appearance of the cyst wall.

Fatal Erysipelothrix rhusiopathiae septicemia in a captive Pacific white-sided dolphin (Lagenorhyncus obliquidens).

One male of a group of seven Pacific white-sided dolphins (Lagenorhynchus obliquidens) died after a brief period of nonspecific clinical signs. Four beluga whales (Delphinapterus leucas) and four harbor seals (Phoca vitulina) were managed in the same water system. Gross examination of the dolphin revealed only moderately enlarged mesenteric lymph nodes. Histopathology revealed small to massive numbers of gram-positive bacilli, usually intravascular, in all tissues. Bacteria were both extracellular and present in macrophages, monocytes, and neutrophils. Aerobic bacterial culture of lung, liver, kidney, and spleen yielded pure cultures of Erysipelothrix rhusiopathiae. Based on clinical course, histopathology, and bacteriology, a diagnosis of acute erysipelas septicemia was made. None of the other cetaceans or pinnipeds exhibited clinical signs.

Small intestinal adeno-like virus in a mountain chameleon (Chameleo montium).

An adult male mountain chameleon (Chameleo montium), one of 92 individuals recently caught in the wild and transported, died after a 28-day history of anorexia. Gross examination revealed marked emaciation and enteric nematodiasis. Histopathologic examination of the small intestine revealed moderate numbers of enterocytes containing 2-15 micrometer-diameter round to ellipsoid, basophilic, intranuclear inclusions. Ultrastructurally, the inclusions consisted of crystalline arrays of hexagonal viral particles 67-76 nm in diameter with electron-dense cores. The viral particles were consistent with an adenovirus. No pathologic changes were associated with the adenoviral infection.

Clinical and clinicopathologic characteristics of ovine GM-1 gangliosidosis.

Ovine GM-1 gangliosidosis is an inherited lysosomal storage disease. Nine lambs affected with the disease were studied to characterize clinical signs and to determine if there were any pathognomonic clinicopathologic abnormalities. Evaluation included physical, ophthalmic, and neurologic examinations, complete blood counts, serum enzyme and electrolyte analyses, urinalyses, cerebrospinal fluid analyses, blood gas analyses, roentgenograms, electromyograms, and electrocardiograms. Two affected lambs had clinicopathologic tests performed before and after the onset of clinical signs. The only consistent abnormalities recognized were nonspecific signs referable to the central nervous system; predominantly ataxia, conscious proprioceptive deficit most severe in the hind limbs, blindness, and recumbency. Lambs continued to eat and drink, though at diminished levels and with loss of body condition. It was concluded that there are no pathognomonic clinicopathologic abnormalities associated with ovine GM-1 gangliosidosis, and antemortem diagnosis requires enzyme assay of leukocytes or cultured fibroblasts, or lectin histochemistry of tissues obtained by biopsy. Lysosomal storage diseases should be considered among the differential diagnoses in young animals presenting with early neonatal death or with nonspecific neurological signs, in concert with an absence of diagnostic clinicopathologic findings.

Ultrastructural lesions of ovine GM1 gangliosidosis.

Ovine GM1 gangliosidosis, an inherited disease of sheep with deficiencies of beta-galactosidase and alpha-neuraminidase, storage of GM1 ganglioside, asialo-GM1 and neutral long chain oligosaccharides in the brain, autosomal recessive inheritance, and histopathologic lesions typical of lysosomal storage diseases, has been described recently. Selected tissues from two sheep with the condition and an age-matched control were examined by transmission electron microscopy to characterize the ultrastructural lesions. In all central and peripheral neurons, the majority of the cytoplasmic space was occupied by membrane-limited enlarged bodies judged to be lysosomes, with a resultant displacement of normal organelles. The neuronal lysosomes usually contained stacks and concentric whorls of lamellae of stored material with a periodicity of 25 to 75 nM. Individual lamellae consisted of fine, multilayered (three to 10, and occasionally more) bands. Less commonly, enlarged neuronal lysosomes contained fibrillogranular or electron dense material. Central nervous system microglia and peripheral nervous system satellite cells had less extensive storage of similar material within enlarged lysosomes, whereas oligodendrocytes, astrocytes, and Schwann cells were relatively unaffected. Hepatocytes and renal epithelial cells also had storage of less quantity than neurons, but within even larger lysosomes. In contrast to neuronal storage material, visceral storage consisted of vesicles containing fibrillogranular or electron dense material within a mostly electron lucent matrix with only occasional lamellae. Kupffer cells and macrophages from bone marrow were affected similarly to but less severely than hepatocytes and renal epithelial cells, whereas hematopoietic cells and chondrocytes were unaffected. Both neuronal and visceral storage were evident, but the neuronal storage was much more extensive.(ABSTRACT TRUNCATED AT 250 WORDS)

Prenatal lesions in an ovine fetus with GM1 gangliosidosis.

Sheep affected with ovine GM1 gangliosidosis are normal at birth and develop clinical signs, initially ataxia, commencing at approximately 5 months of age, which progresses rapidly to recumbency. Superovulation and embryo transfer techniques were applied to a flock of carrier sheep of ovine GM1 gangliosidosis to increase the numbers of carrier and affected animals. A recipient ewe with 3 at-risk fetuses died at 4 months of gestation (normal ovine gestation is 5 months), and spectrofluorimetric assay of cerebral lysosomal beta-galactosidase of the fetuses showed that 2 were carriers and one was an affected fetus. The affected fetus had marked cytoplasmic enlargement and vacuolization of central and peripheral nervous system neuronal soma and of hepatocytes and renal epithelial cells. Lectin histochemistry indicated abnormal storage of complex carbohydrates, with terminal saccharide moieties consisting of beta-galactose, N-acetylneuraminic acid, and N-acetylgalactosamine. This case underlines the need for prenatal initiation of therapy and also demonstrates that vacuolization alone is not the cause of clinical signs in this lysosomal storage disease in that clinical signs do not commence until at least 5 months after vacuolization is histologically apparent.

Inheritance of an ovine lysosomal storage disease associated with deficiencies of beta-galactosidase and alpha-neuraminidase.

Prospective and retrospective genetic studies were performed on sheep with a recently described inherited lysosomal storage disease that involves a profound deficiency of beta-galactosidase and an associated deficiency of alpha-neuraminidase. Retrospective studies of the flock of sheep in which four affected lambs were born indicated little inbreeding but the presence of a common ram in both the maternal and paternal sides of the pedigrees. When unrelated rams were used in the flock in subsequent years, no affected lambs were born. The affected lambs' parents were phenotypically normal, so the disease was investigated as a putative autosomal recessive condition in prospective breedings of related sheep over two breeding seasons. For the third breeding season, heterozygous ewes were superovulated and bred to a heterozygous ram, and the resultant embryos were transferred to recipient ewes. Later in the same breeding season, the heterozygous ewes were re-bred naturally to the heterozygous ram. Lambs were identified as affected by the development of signs of ataxia, levels of beta-galactosidase that were less than 7% of the levels in controls by spectrofluorometric assay, or the histopathologic demonstration of vacuolization of neurons. Heterozygous sheep were identified by the production of affected offspring and/or by levels of beta-galactosidase in fibroblast cultures that were approximately 50% of control levels. The phenotypic ratio of affected sheep to normal sheep and the genotypic ratio of affected to heterozygous to normal sheep were consistent, by chi-square analysis, with an autosomal recessive trait. It was concluded that this ovine lysosomal storage disease is an autosomal recessive disease.

Expression of beta-galactosidase in preimplantation ovine and porcine embryos.

Knowledge regarding the timing of embryonic expression of the mammalian genome is of relevance for the development of preimplantation diagnostic methods for human genetic diseases. For development of preimplantation diagnosis of lysosomal storage diseases, it will be necessary to know at which embryonic stage the genes for lysosomal enzymes are expressed. In previous studies by other investigators, it has been shown that lysosomal alpha- and beta-galactosidase and beta-glucuronidase in murine embryos increase 50- to 100-fold in activity between the two-cell and late blastocyst stage. We describe here expression of lysosomal beta-galactosidase in preimplantation ovine (two-cell through midblastocyst) and porcine (two-cell through late blastocyst) embryos. Expression of beta-galactosidase in ovine and porcine preimplantation embryos followed a similar rate of increase as that described for murine embryos. Activity of beta-galactosidase increased over 10-fold between the two- to four-cell and midblastocyst stages in ovine embryos, and 300-fold between the two- to four-cell and late blastocyst stages in porcine embryos. Activity expressed on a per cell basis was relatively constant in ovine embryos, as has been described in murine embryos, and increased approximately 5-fold on a per cell basis in porcine embryos. Activity of beta-galactosidase in ovine and porcine embryos initially was greater than 12-fold on a per cell or per embryo basis than in murine embryos evaluated. The knowledge of beta-galactosidase embryonic expression may provide the basis for preimplantation diagnosis of genetic beta-galactosidase deficiency in these species.

Interspecific genetic complementation analysis of human and sheep fibroblasts with beta-galactosidase deficiency.

Interspecific somatic cell hybrids were analyzed by genetic complementation to determine if a lysosomal storage disease in sheep associated with deficiencies of beta-galactosidase and alpha-neuraminidase was homologous with any of four beta-galactosidase-deficient human diseases. Fibroblasts from beta-galactosidase-deficient sheep, cats, and human patients were fused and assayed histochemically for beta-galactosidase, with 5-bromo-4-chloro-3-indolyl beta-D-galactoside. We observed complementation in heterokaryons consisting of fibroblasts from beta-galactosidase-deficient sheep and fibroblasts from patients with galactosialidosis or mucolipidosis type II, but no complementation in heterokaryons consisting of fibroblasts from beta-galactosidase-deficient sheep and fibroblasts from human or feline GM1 gangliosidosis (type I) or from human mucopolysaccharidosis type IVB fibroblasts. We conclude that the ovine disease is due to a mutation at the genetic locus homologous with that of GM1 gangliosidosis and mucopolysaccharidosis type IVB, suggesting that the primary defect in the ovine disease is a mutation of the beta-galactosidase structural gene.

Lectin histochemistry of an ovine lysosomal storage disease with deficiencies of beta-galactosidase and alpha-neuraminidase.

Lectin histochemistry is a useful technique to identify and to localize in cells and tissues the terminal carbohydrate moieties of glycoproteins and glycolipids. The specific diagnosis of some glycoprotein storage diseases was accomplished using lectin staining patterns, and such methods of diagnosis have been attempted for some glycolipid storage diseases. This technique was applied to formalin-fixed paraffin-embedded and frozen neural, hepatic, and renal tissues of sheep with an inherited lysosomal storage disease with deficiencies of beta-galactosidase and alpha-neuraminidase. The cytoplasm of central nervous system neurons of affected sheep in paraffin-embedded sections stained with peanut agglutinin (PNA), Ricinus communis agglutinin-I (RCA-I), Dolichos biflorus agglutinin (DBA), and soybean agglutinin (SBA). The cytoplasm of neurons in frozen sections of these tissues stained with PNA, RCA-I, wheat germ agglutinin (WGA), and Ulex europaeus agglutinin-I (UEA-I). The cytoplasm of frozen and paraffin-embedded sections of liver and kidney of affected sheep stained with PNA, whereas paraffin-embedded sections also stained with RCA-I. These results suggest the stored material in this disease has terminal saccharide moieties consisting of beta-galactose, N-acetylneuraminic acid, and N-acetylgalactosamine. Paraffin processing altered lectin staining patterns. Although the staining pattern in this glycolipid storage disease was complex, lectin histochemistry may prove to be a useful technique for the characterization of storage products and for the diagnosis of lysosomal storage diseases.

The lesions of an ovine lysosomal storage disease. Initial characterization.

An inherited disease associated with deficiencies of beta-galactosidase and alpha-neuraminidase has been identified recently in sheep. The clinical signs, the deficiency of lysosomal enzymes, and the familial nature of the disorder suggested that the condition was a lysosomal storage disease. Four affected sheep were necropsied and their tissues were examined by histopathologic and histochemical methods to determine if the lesions were consistent with a lysosomal storage disease. Central nervous system neurons were enlarged with finely to coarsely granular cytoplasmic material, or less often, neurons were distended with multiple, variably-sized vacuoles. Loss of neurons without gliosis was evident and the Nissl substance was either dispersed and fragmented or condensed around the nuclei of remaining neurons. Neurons of intestinal and other peripheral ganglia, retinal ganglion cells, and heart Purkinje fibers were enlarged similarly. White matter of the cerebrum and spinal cord had numerous spheroid to ellipsoid axonal enlargements. Periportal hepatocytes and renal epithelial cells were enlarged with marked vacuolation. The neuronal storage material stained intensely with periodic acid-Schiff/alcian blue, with Luxol fast blue, for acid phosphatase, and moderately with oil red O stains. Renal and hepatocyte storage material stained intensely with oil red O and moderately with periodic acid-Schiff/alcian blue and Sudan black B stains. The lesions in these sheep were consistent with those of a lysosomal storage disease. Both neuronal and visceral storage occurred, but the neuronal storage was more severe.

Beta-galactosidase activity in fibroblasts and tissues from sheep with a lysosomal storage disease.

Tissues and fibroblasts of sheep affected with an inherited, neuronal lysosomal storage disease expressed a deficiency of beta-galactosidase activity. Cerebrum, kidney, lung, spinal cord, and spleen from affected sheep had less than 8% of the beta-galactosidase activity present in the respective tissues of normal sheep. No evidence for the presence of an endogenous inhibitor in affected sheep was detected by mixing studies. Liver of affected sheep expressed a deficiency of beta-galactosidase activity only in the presence of the beta-D-glycosidase inhibitors, glucono-delta-lactone and 2,5-dihydroxymethyl-3,4-dihydroxypyrrolidine. In these studies, we demonstrated the existence of tissue-specific beta-galactosidases in sheep and showed that the affected sheep have a deficiency of the lysosomal beta-galactosidase. Our results suggest that the high residual beta-galactosidase activity in liver of affected sheep can be attributed to a nonlysosomal beta-galactosidase that has a neutral pH optimum and may be under temporal regulation.

Inherited lysosomal storage disease associated with deficiencies of beta-galactosidase and alpha-neuraminidase in sheep.

Histopathologic, ultrastructural and Golgi impregnation studies disclosed lesions characteristic of a neuronal lysosomal storage disease in related sheep with onset of neurologic signs at 4-6 months. Biochemical and enzymatic evaluation disclosed storage of GM1 ganglioside, asialo-GM1, and neutral long chain oligosaccharides in brain, urinary excretion of neutral long chain oligosaccharides, and deficiencies of lysosomal beta-galactosidase and alpha-neuraminidase. Retrospective and limited prospective genetic studies suggested autosomal recessive inheritance. A gene-dosage effect on beta-galactosidase levels was documented in fibroblasts from putative heterozygous sheep. Fibroblasts from affected sheep did not have increased beta-galactosidase activity after incubation with the protease inhibitor, leupeptin. In some aspects this disease is similar to GM1 gangliosidosis, but is unique in that a genetic defect in lysosomal beta-galactosidase may cause the deficiency of lysosomal alpha-neuraminidase.

Visceral gout in a rough legged hawk (Buteo lagopus).

Visceral gout is reported for the first time in a rough legged hawk (Buteo lagopus). Urate crystals were present on the pericardium, thoracic and abdominal air sacs, and the ventral surface of the liver. The liver and spleen also had urate crystals throughout the parenchyma. There was no indication of articular or renal involvement. The immediate cause of death in this hawk was not identified, but appeared to result from multiple factors, including the visceral gout.

Peribronchiolar fibrosis in lungs of cats chronically exposed to diesel exhaust.

This study reports the quantitative changes in the pulmonary proximal acinar region following chronic exposure to diesel exhaust and following an additional 6 months in clean air. Cats (13 months of age) from a minimum disease colony were exposed to clean air (eight cats for 27 months and nine cats for 33 months), diesel exhaust for 8 hours/day, 7 days/week (nine cats for 27 months), or diesel exhaust for 27 months followed by 6 months in clean air (10 cats). Morphologic and morphometric evaluation using light microscopy and scanning and transmission electron microscopy revealed two major exposure-related lesions in proximal acinar regions of lungs of cats: peribronchiolar fibrosis associated with significant increases in lymphocytes, fibroblasts, and interstitial macrophages containing diesel particulate-like inclusions and bronchiolar epithelial metaplasia associated with the presence of ciliated and basal cells and alveolar macrophages containing diesel particulate-like inclusions. Peribronchiolar fibrosis was greater at the end of the 6 months in clean air following exposure, whereas the bronchiolar epithelial metaplasia was most severe at the end of exposure. Following an additional 6 months in clean air the epithelium more closely resembled the control epithelial cell population. The labeling index of terminal bronchiolar epithelium was significantly increased at the end of exposure but was not significantly different from controls or exposed cats following an additional 6 months in clean air. The ultrastructural appearance of epithelial cells remained relatively unchanged following diesel exhaust exposure with the exception of diesel particulate-like inclusions. Total lung collagen, expressed as hydroxyproline per left caudal lobe, was apparently increased (although the difference was not significant) in lungs of cats allowed to recover 6 months in clean air. Newly synthesized collagen (evaluated as the amount of cross-link-derived aldehydes in collagen) was significantly increased to more than twice the control values. The ratio of collagen aldehydes to hydroxyproline was also significantly increased. These observations imply that chronic exposure to diesel exhaust has a persistent fibrogenic effect on the proximal acinar region of the lung.