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Ovine GM1 gangliosidosis is a newly described neuronal lysosomal storage disease first observed in Suffolk sheep. Affected sheep are clinically normal at birth and exhibit ataxia at 4 to 6 months of age which rapidly progresses to prostration. Affected sheep exhibit a profound deficiency of lysosomal β-galactosidase in fibroblasts and tissues, and a partial deficiency of α-neuraminidase. Abnormal accumulation of GM1 ganglioside, asialo-GM1 and neutral long chain oligosaccharides is present in the cerebrum with excretion of neutral long chain oligosaccharides in the urine. Genetic studies indicate the disease is inherited as an autosomal recessive. Pathologic lesions consist of marked distension and vacuolation of the cytoplasm in virtually all neurons (central and peripheral nervous system) and of periportal hepatocytes and renal epithelial cells. Preliminary ultrastructural studies disclosed numerous membranous whorls and lamellar stacks within membrane-bound vacuoles in neurons. Golgi staining of cerebrum discloses formation of spiny meganeurities and secondary neurite formation in pyramidal neurons. Lectin histochemistry indicates stored material has terminal saccharide moieties consisting of β-galactose, N-acetylneuraminic acid, and N-acetylgalactosamine. No consistent clinicopathologic and no musculoskeletal abnormalities have been identified. The prevalence and economic significance of ovine GM1 gangliosidosis is unknown. Most neonatal deaths and abortions of small ruminants remain undiagnosed, and possibly many have an undetermined genetic basis. The described disease emphasizes the importance of clinical and pathologic evaluation of neonatal deaths as the economic and research importance of genetic diseases are unrealized.

Major alterations are induced in muscle cells infected by either Trichinella spiralis or Trichinella pseudospiralis. To investigate the response of muscle to these infections we have analyzed the expression of acid phosphatase (ACP, EC 3.1.3.2), adult skeletal muscle myosin heavy chain, and muscle tropomyosin proteins in infected mouse skeletal muscle cells. Using T. spiralis-infected cells, we provide strong evidence that the tartrate-sensitive ACP of these cells was synthesized by the infected cell and localized in lysosomes. Isoenzyme analysis indicated that the ACP activity was of host muscle cell origin and the specific activity of this ACP was 2.5 times greater than that in associated inflammatory cells. Increased ACP activity was also demonstrated in muscle cells infected by T. pseudospiralis. In synchronized muscle infections, increased ACP activity was detected at 5 days post-muscle infection for both parasites. ACP activity was further increased in infected muscle cells at later times tested. This increased infected cell ACP activity represents the earliest positive enzyme marker yet described indicating expression of the infected cell phenotype. In contrast, myofibrillar proteins were not detected in muscle cells chronically infected by T. spiralis but were detected in muscle cells infected by T. pseudospiralis. Decrease in myofibrillar protein levels was detected by 10 days post-muscle infection by T. spiralis. The data presented demonstrate significant differences and similarities in the phenotypes of muscle cells infected by these two parasites and establish criteria that could facilitate identification of parasite factors that may be involved in these phenomena.

SummaryThe ultrastructure of lysosomes from bovine megakaryocytes (MK) and platelets was characterized using acid phosphatase cytochemistry with beta-glycerophosphate as substrate and cerium as a trapping agent. The technique was easily reproducible; cerium-phosphate precipitates were uniform, readily visualized, and there was a virtual absence of nonspecific reaction product. Acid phosphatase was localized in the trans aspect of the Golgi complex and/or granules of less than 50 nm to 650 nm diameters in MK at all stages of maturation. Forty percent of the MK lysosomes contained inclusions of variable shapes, sizes and electron-density and were classified as secondary lysosomes. Twenty-four percent of the platelet sections contained acid phosphatase-positive granules. Fifty-four percent of these were secondary lysosomes. This is the initial report demonstrating secondary lysosomes in either resting MK or platelets using acid phosphatase cytochemistry. These findings suggest that MK and platelet lysosomes have an intracellular function in resting MK and platelets.

SummaryThe ultrastructure of lysosomes from megakaryocytes (MK) and platelets of cattle with the Chediak-Higashi syndrome (CHS) was characterized using acid phosphatase histochemistry with beta-glycerophosphate as substrate and cerium as a capturing agent. Acid phosphatase was localized in the trans aspect of the Golgi complex and/or granules in MK at all stages of maturation. Morphometric analysis of the diameter of each lysosome was performed on MK from CHS cattle and compared to MK from normal cattle. Lysosomes in CHS MK were neither enlarged nor different with respect to classification as secondary lysosomes, which composed 35% of the lysosomes in CHS MK. Lysosomes were demonstrated in 22% of the CHS platelet sections and appeared similar to those from normal cattle, 56% of them being classified as secondary lysosomes. Why lysosomes are not enlarged in bovine CHS MK and platelets, whereas they are enlarged in most other cell types, remains unknown.

We evaluated four canine cutaneous mast cell tumors cytogenetically. All four tumors contained both hypodiploid and hyperdiploid cells, an increase in the number of metacentric chromosomes, exchange configurations, and cells showing loss of an X chromosome. All tumors contained metaphases with chromosome gaps and breaks at frequencies greater than observed spontaneous chromosome breaks in normal cultured canine peripheral blood lymphocytes. Three of the four tumors had a normal modal chromosome number of 78. The fourth tumor had a modal chromosome number of 93, which represented 15% of the cells evaluated from this tumor.

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.

Young rabbits treated with multiple intravenous injections of Adriamycin (doxorubicin hydrochloride), a broad-spectrum antitumor antibiotic, over a period of 3–5 months develop an unthrifty appearance associated with decreased gains in body weight. A dose schedule of 0.7 mg/kg (7.7 mg/m2 body surface area) three times per week produces lesions consistently over a period of 13–18 weeks at cumulative doses of 27.3 mg/kg (300 mg/m2) - 37.8 mg/kg (416 mg/m2), respectively. Roentgenographs of long bones reveal thin cortices and narrowed diaphyseal shafts (Figs. 363,364). Epiphyseal areas have a generlized radiolucent appearance with lytic patterns varying from motheaten (cancellous type) to cystic. Multiple lucent holes of varying sizes are scattered throughout the epiphysis intermixed with prominent trabecular densities. Metaphyseal zones frequently contain radiodense coarse trabeculation representative of tightly packed cancellous bone (Fig. 364).

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.

Analysis of breeding data revealed that the muscular dystrophy trait in a pedigree of mink is transmitted in an autosomal recessive manner. Variation in skeletal muscle fiber diameter size is the most pronounced and consistent change in the dystrophic mink. Other changes include centralization of nuclei, degenerative change, increase in endomysial and perimysial connective tissue, and regenerative attempts. These changes are not present in known heterozygotes.

Although the autosomal recessive disease Chediak-Higashi syndrome (CHS) has been described in humans, cats, mink, cattle, mice, killer whales, blue foxes, and silver foxes, and these conditions appear quite similar, no direct evidence of the homology of this disease in the various species has been presented. To determine if CHS in humans, cats, mink, cattle, and mice is due to a mutant gene at the homologous genetic locus in each species, or alternatively, if these are merely similar syndromes, genetic complementation analysis after interspecific somatic cell (fibroblast) hybridization was performed. "Paracrystal" formation was the criterion used for the determination of complementation. The initial studies in this report were designed to characterize paracrystal formation in control and CHS fibroblasts of these five species. Most of the control fibroblasts from each species (91-96.6%) formed paracrystals upon incubation with 25 micrograms/ml of the microtubule depolymerizing agent vinblastine sulfate. A significantly (P less than 0.05) smaller percentage of the CHS fibroblasts formed paracrystals after the same incubation (except CHS mice, with 90.2% paracrystals). It was found that 52% of the human CHS fibroblasts, 60% of cat CHS fibroblasts, 47% of mink CHS fibroblasts, and 53.8% of cow CHS fibroblasts formed paracrystals. For genetic complementation analysis, human CHS fibroblasts were fused to cat, mink, cow, or mouse CHS fibroblasts with polyethylene glycol. Control fusions were human CHS fibroblasts fused with human, cat, mink, cow, and mouse normal fibroblasts. The results of complementation analysis after the fusion of human CHS with cow CHS and human CHS with mouse CHS fibroblasts were inconclusive. A lack of complementation of human CHS with cat CHS and human CHS with mink CHS fibroblasts indicates that the disease is homologous in these species.

The chicken embryo is a useful animal model for investigating problems in developmental biology and teratology. Here we report data that further define the causes of 2 different patterns of malformation (one associated with amnion abnormalities, the other with isolated neural tube defects) and death induced by making a window in the shell and subshell membranes during the first day of incubation. The interpretation of these data suggests to us the following hypotheses. An early amnion deficit spectrum or syndrome (EADS) in chicken embryos is caused by a brief (less than 10 sec) perturbation that occurs during the windowing procedure. This perturbation results in an acute increase in mechanical tension to the developing embryo and support structures, dehydration localized to the area of the blastoderm, and/or increased friction between the blastoderm and overlying vitelline and shell membranes. Isolated neural tube defects (NTDs) are caused by a longer perturbation (greater than 3 hr) consisting of increased mechanical stress across the blastoderm. The mechanical stress is associated with the introduction of a new air space over the animal pole of the yolk during windowing. The new air space causes the shape of the yolk to change (ie, to be deformed), resulting in an increase in mechanical tension across the vitelline membrane and blastoderm. NTDs involving the head are associated with significant early embryonic mortality, whereas those involving the trunk are not. Death may also be caused by cardiovascular anomalies observed in EADS. It is concluded that disturbances in morphogenesis and death in this model are, therefore, the result of extrinsic forces (eg, mechanical stress, localized dehydration, or friction) acting on different tissue types at various critical times in development. Intensity and duration of these forces on the developing blastoderm are important variables.

Previous reports suggest that windowing the shells of chicken eggs during the first day of incubation frequently results in dysmorphogenesis of the central nervous system. We report here data that further delineate the neural tube defects associated with this animal model. In addition, we describe another birth defect syndrome associated with windowing: the early amnion deficit spectrum (EADS). Several components of the egg are altered structurally by windowing: the shell, outer and inner shell membranes, yolk, and air space at the blunt end of the egg. A new air space is formed over the embryo as the original one at the blunt end is obliterated. A series of studies (pH, oxygen and carbon dioxide tensions, relative humidity, temperature, and deformation of the yolk documented with magnetic resonance imaging) examining individual steps of the windowing procedure and additional techniques that stimulate windowing suggest that mechanical stress causes isolated neural tube defects and dehydration causes amnion defects. These amnion defects are associated with other embryonic abnormalities suggestive of deformations consistent with EADS.

Ceroid-lipofuscinosis is described in Australian Cattle dogs. Lesions included storage of ceroid-lipofuscin in most tissues with characteristic ultrastructural inclusion body patterns in neurons and other cells. Dolichol concentration of the affected dog's brain was similar to those in age-matched control dog brains. However, concentrations in brain, liver and kidneys were markedly higher than in a slightly younger dog. Biochemical data including lysosomal enzyme activities exclude other lysosomal storage diseases. The clinical and pathological features of this disorder resemble those of the juvenile Spielmeyer-Vogt type of Batten disease in humans.

Tissues and fibroblasts of sheep affected with an inherited, neuronal lysosomal storage disease expressed a deficiency of β-galactosidase activity. Cerebrum, kidney, lung, spinal cord, and spleen from affected sheep had less than 8% of the β-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 β-galactosidase activity only in the presence of the β-d-glycosidase inhibitors, glucono-δ-lactone and 2,5-dihydroxymethyl-3,4-dihydroxypyrrolidine. In these studies, we demonstrated the existence of tissue-specific β-galactosidases in sheep and showed that the affected sheep have a deficiency of the lysosomal β-galactosidase. Our results suggest that the high residual β-galactosidase activity in liver of affected sheep can be attributed to a nonlysosomal β-galactosidase that has a neutralpH optimum and may be under temporal regulation.

Craniomandibular osteopathy is a disease of several breeds of dogs, principally West Highland White and Scottish terriers. It is characterized by a non-neoplastic proliferation of bone on the ramus of the mandible and/or the tympanic bulla. The disease in various respects resembles Paget's disease and infantile cortical hyperostosis of humans. A retrospective pedigree analysis of a kindred of West Highland White terriers was performed to determine if the trait was inherited and to determine mode of inheritance. This study indicated that in West Highland White terriers, the condition is an autosomal recessive trait.

The relation between the rabbit gastrointestinal (colonic) isozyme of lysozyme and the unique circadial coprophagic behavior of rabbits, termed "cecotrophy," was investigated. We found that lysozyme activity was very low in all segments of the rabbit gastrointestinal tract proximal to the fusus coli. However, the distal or nonsacculated colon, located distal to the fusus coli, had very high lysozyme activity throughout. It was determined, furthermore, that the lysozyme in the wall of the nonsacculated colon was secreted circadially into the colonic lumen in association with the circadial production of soft feces that were destined for reingestion by cecotrophy. The colonic lysozyme, therefore, was transferred to the stomach. These observations suggest that rabbit colonic lysozyme may have a possible function in the upper gastrointestinal tract and be analogous to ruminant abomasal-duodenal lysozyme.

A group of related borzoi dogs were studied over a 6-year period to ascertain the cause of primary hypothyroidism. Four generations of dogs were analyzed. Two littermates with lymphocytic thyroiditis were mated and the 10 offspring were all diagnosed, on the basis of thyroid biopsy evaluation, as having lymphocytic thyroiditis by age 2.5 years. A wide range of thyroid gland lesions was demonstrated in this litter of dogs. This report documents the occurrence of lymphocytic thyroiditis in three successive generations of an inbred group of borzoi dogs. An autosomal recessive mode of inheritance for the trait in this group of dogs is proposed.

A choroid papilloma in the choroid plexus of the ala of the fourth ventricle in a mature male elasmobranch, Squalus acanthias, was described. This is apparently the first report of a neoplasm of the central nervous system in a member of the class Chondrichthyes.

Although natural killer (NK) cells are thought to give the host a spontaneous resistance against tumours and have been postulated to act in vivo as surveillor cells, definitive data in support of these hypotheses has not been obtained. Recently the beige (bg) mouse, a morphological homologue of the human Chediak-Higashi (CH) syndrome, was shown to be deficient in NK activity. Specifically, spleen cells of bg mice were demonstrated to be incapable of in vitro natural cytotoxicity against tumour cells. We report here that a tumour line, modified to be sensitive to NK cytotoxicity by in vitro culture, demonstrated in vivo an increased growth rate, faster induction time and an increased metastatic capability in bg compared to control mice. This was not found with a tumour line insensitive to NK activity (without in vitro culture). In vivo activation of NK cells in bg and control mice resulted in a decrease in tumour growth rate and metastatic frequency. These results demonstrate that NK cells have an important function in the host's control of tumour growth and metastasis.

High levels of gastrointestinal lysozyme were present in the stomach of guinea pigs, but not in other portions of the gastrointestinal tract. Because the cecum is the fermentation organ of guinea pigs, these observations call into question the validity of the current hypothesis that the gastrointestinal lysozyme of herbivores functions in the digestion of bacteria from the anterior fermentation organ.

The role of natural killer (NK) cells in tumor growth and metastasis was studied in syngeneic normal and beige inbred C57BL/6 mice. Mice with the beige point mutation have been shown to be deficient in nonstimulated NK activity. Tumor-passaged B16 malignant melanoma cells were refractory to NK activity as determined by in vitro assay, but after in vitro culture they became sensitive to NK activity. The NK-insensitive B16 tumor grew and metastasized similarly in normal and beige mice. However, the NK-sensitive B16 tumors grew more slowly and produced fewer metastases in normal mice than in NK-deficient beige mice. Activation of NK cells by lymphocytic choriomeningitis virus infection decreased the rate of growth and number of metastases of both NK-sensitive and NK-insensitive tumors in both normal and beige mice. These results suggest the importance of NK cells as a determinant of tumor growth and metastasis.

The Chediak-Higashi syndrome (CHS) is a genetic disorder of man, cats, and four other animal species. Enlarged cytoplasmic granules, including lysosomes and melanosomes, characterize the syndrome. Cats affected with CHS lack funduscopically visible tapeta. In normal cats, the tapetum is the light reflecting cellular layer located in the choroid. The tapetal cells contain bundles of parallel cytoplasmic rods. In this study, eyes from CHS and control cats were examined by light microscopy and transmission electron microscopy. The CHS kittens up to 14 days of age had tapeta which appeared similar to those of the controls. By 28 days of age some of the CHS tapetal rods had degenerated. Degeneration of the tapetal rods progressed rapidly and by 56 days of age there was a dramatic difference in the ultrastructural appearance of the tapetal cells. All the rods had degenerated and the contents of the tapetal cells were disorganized. The tapetal layer gradually thinned over a period of several months until the layer was absent or nearly so in CHS cats over one year of age. This study demonstrated that there is a previously overlooked degenerative component of the Chediak-Higashi syndrome.

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.

Fawn-hooded (FH) rats were shown to lack the genetically conditioned defect of natural killer (NK) activity hypothesized to be present by analogy with the Chediak-Higashi syndrome (CHS) in mice and human beings. In 4-h 51Cr release assays, splenic NK cells from FH rats killed YAC-1, RL male l and G1-TC tumor targets without deficiency based upon comparison with cells from BD-IV, BD-IX and NBR inbred rat strains. Progeny of BD X FH F1 rats backcrossed to FH failed to reveal a correlation of reduced NK activity and dilute coat color. From these, and other data presented, it is concluded that despite similarities in coat color dilution and platelet storage pool deficiency. FH rats do not closely resemble CHS mice or human beings in having deficient NK activity and cannot be considered the rat homolog of the CHS.

Familial canine dermatomyositis, an inflammatory disease of skin, muscle, and sometimes vessels, was first reported in 1984. 1,2 Canine dermatomyositis (DM) has been recognized only in collies and Shetland sheep dogs (Shelties). 1,3,4 The condition in collies, diagnosed in dogs in 19 states in the United States, is well documented and widespread. 5 The condition in Shelties is less well characterized. 3,4 Dermatomyositis develops spontaneously in juvenile dogs as a variably severe dermatitis in the skin of the face, ears, distal extremities over bony prominences, and tip of the tail. 1,6 Myositis develops later, principally in muscles of mastication and muscles of extremities below the elbow and stifle. 7,8 Severely affected dogs have more generalized dermatitis and myositis. 7,8

In this study designed to characterize the renal lesions in cats with the autosomal recessive Chediak-Higashi-Steinbrinck syndrome (CHS), the renal tubular epithelial cells of CHS cats were examined by light microscopy. Numerous cells of proximal convoluted and straight tubules, thin loop of Henle, distal convoluted and straight tubules, and collecting tubules contained enlarged cytoplasmic granules morphologically consistent with lysosomes. In general, the enlarged lysosomes were larger and more numerous in proximal convoluted and straight tubular cells and were generally more massive in older cats. The lesions observed were similar to those in the renal epithelial cells of other species with CHS and were consistent with those reported previously in other tissues of CHS cats. It is concluded that CHS cats are an appropriate model in which to study the effects of the CHS renal lesions on renal function in this syndrome.