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Chediak-Higashi Syndrome (CHS) is a well-characterized, autosomal recessively inherited lysosomal disease caused by mutations in lysosomal trafficking regulator (LYST). The feline model for CHS was originally maintained for ~20 years. However, the colonies were disbanded and the CHS cat model was lost to the research community before the causative mutation was identified. To resurrect the cat model, semen was collected and cryopreserved from a lone, fertile, CHS carrier male. Using cryopreserved semen, laparoscopic oviductal artificial insemination was performed on three queens, two queens produced 11 viable kittens. To identify the causative mutation, a fibroblast cell line, derived from an affected cat from the original colony, was whole genome sequenced. Visual inspection of the sequence data identified a candidate causal variant as a ~20 kb tandem duplication within LYST, spanning exons 30 through to 38 (NM_001290242.1:c.8347-2422_9548 + 1749dup). PCR genotyping of the produced offspring demonstrated three individuals inherited the mutant allele from the CHS carrier male. This study demonstrated the successful use of cryopreservation and assisted reproduction to maintain and resurrect biomedical models and has defined the variant causing Chediak-Higashi syndrome in the domestic cat.

A 5-year-old male Labrador Retriever had progressive incoordination, visual impairment, and exercise intolerance. Coarse facial features, macrodactylia, unilateral corneal dystrophy, generalized osteopenia, progressive neurologic deterioration, and a positive urine spot test for acid mucopolysaccharides suggested mucopolysaccharidosis. Intracytoplasmic vacuoles were most prevalent in epithelial cells, endothelial cells, and histiocytes of liver, kidney, thyroid gland, and spleen. Ultrastructural examination disclosed electron-lucent floccular to lamellar membrane-bound storage material characteristic of mucopolysaccharides. Periodic acid-Schiff-positive intracytoplasmic material was identified in multiple neurons in the medulla, pontine nucleus, cerebellum, and spinal gray matter horns. Biochemical assays identified a deficiency in iduronate-2-sulfatase (IDS) activity in cultured dermal fibroblasts compared with normal dogs. Hair root analysis for IDS showed that the dam was a carrier of X-linked Hunter syndrome and that a phenotypically normal male littermate of the affected dog was normal. This is the first report of Hunter syndrome or mucopolysaccharidosis type II in a dog.

Lysosom al storage diseases are caused by inherited defigangliosidos is has recentl y been characterized." A comparciencies of vario us lysosomal enzymes and result in the proati ve study oflectin bind ing pattern s was per formed on cengress ive sto rage of uncatab olizabl e substrate .v'v Th e specific tral nervou s system tissue from two lambs affected with ovi ne enzyme deficiency(ies) and residual activ ity of the deficient GM , gangliosidos is and a lam b affected with the disease seen enzy me(s) for natural subs trates determ ine which orga n is in New Zealand. (organs are) pr im aril y affected and, thu s, the clini cal presenLect in hi st och emi st ry was co nd uc ted as pr evi ou sly tati on of eac h lysosom al storage disease. T hey are usually described! on formalin-fixed and paraffin-embedd ed sectio ns inh erited as autosom al recessive disord ers and often result of cerebru m, cerebellum, and brain stem or spinal cord on in death du ring childhood. Th ere are ove r 30 human Iysothe following: fro m an 8-mo nth-o ld lamb (tissues in a 28soma l sto rage diseases, and an imal models of these di seases year-o ld paraffin block) affected with the disease seen in New represent a valuable reso urce for the development of new Zea land (New Zealand lamb); two 6-mo nth-old lambs afdiagnostics and therap euti cs and for furt her elucida tio n of fected with ov ine G M , gangliosidosis; and a 6-month-old lysosomal functio n. normal (contro l) lamb. Th e biotinylated lect ins and concenWe have recently descr ibed an autosoma l recessive lysotrat ion s used , maj or carbohydra te specificities of each lectin , soma l storage dise ase in Suffolk sheep with a pro found deand the binding inh ibit ors used are listed in Table I . Negati ve ficiency of lysosomal {1-galactos idase and partial deficiency contro ls consisted of sections incub ated with phosphate buffofa -neuram inidase that result s in the death of affected lambs ered saline alone instead oflectins. Incub at ion of eac h lectin at 4 to 6 mon th s of age.v" Th ough there is a dual enzyme with its correspo nd ing binding inhibito r before applicatio n deficiency, thi s ovi ne disease is homo logous with human to a match ing set of slides served as con tro ls for binding G M, gangliosidosis (deficie ncy of{1-galactosidase) and is not specificity . hom ologou s with human galactosia lidos is (de ficiencies of T he lectin binding pattern ofcen tra l nervous system tissue {1-galactosidase and a -neuraminidase ) based upon interspefrom the two lambs affected with ovi ne GM, gangliosido sis cific genetic complementation analysis of somatic cell hywas the same as previou sly describ ed' and was present in all brids. ' The disease has thus been term ed ov ine G M) ganareas of the central nervou s system. Th e cytoplasm of neugliosidosis. A simi lar di sease was seen in a gro up of relat ed rons (including Purkinje, Go lgi, and stellate basket cells and 8-month-o ld lambs on one farm in New Zealand ov er 28 cells of the mo lecular layer of the cere bellum) stai ned with years ago an d termed, " lipid-like neuronal degenerati on. " ? peanut (Arachis hypogea) agglutinin (PNA ), castor bean (RicAs with ovi ne G M , ganglios idosis, lambs suddenly develinus com m unis) agglutinin-I (RCA-I), horse gram (Dolichos oped atax ia that rapidly progressed to prostration . Lesion s bijlorus) agglutinin (DBA), soybea n (Glycine max ) agglut inin in the centra l nervous system were character istic of a neu(SBA), and horseshoe crab (Limulus polyphemus) agglutinin ronal lysosom al storage disease: there was diffuse enlargeto a substantia lly great er degree than the neuronal cytoplasm ment and vac uolatio n of virtually all neuron s; and the lesions of contro l sheep. Th e central nervou s system tissue of the were identical to those seen in ovi ne G M , gangliosidosis New Zealand lamb had a nearl y identica l lectin bind ing pat(Figs. I , 2). tern as that observe d in the two lamb s affected with ovi ne Lectin histochemi stry has been used for the charact erizaG M , gangliosidosis (Figs. 3-8). Th e onl y differences were tion of sto red products in many lysosom al sto rage diseases. stro ng binding of LPA by neuron s from the New Zealand Lectin s are agglut inating proteins with well-characterized and lamb versus slight binding by neuron s from the two lambs specific affi nities for carbo hydrates and, when bound to visaffected with ovi ne G M , gangliosidos is, and slight to negative ualant s, allow localizati on and ident ificat ion of carbohybind ing of WGA by neurons fro m the New Zealand lamb, drat es in tissues. Unique lecti n bindi ng pattern s have been vers us stro ng to slight binding of WGA by neuro ns from the observed for different lysosom al sto rage diseases, and simi lar two lambs affected with ovi ne G M ) gangliosidosis. Incubapatterns have been observed for the same sto rage disease in tion of each lectin with its corresponding inhibitor consisd ifferent species. Th e lectin bind ing pattern of ovi ne G M , tentl y decreased or blocked bind ing of the lectin to tissue

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)

The autosomal recessive disease Chediak-Higashi syndrome (CHS) is a progressive and generally fatal disease of humans. The underlying genetic defect in CHS is unknown and prenatal diagnostic methods have not been applied to this disease. The purpose of this study was to determine if CHS chorionic cells expressed a characteristic of CHS--enlarged lysosomes--that would permit the prenatal diagnosis of the disease. Cats with CHS, which have been shown to be homologous with human CHS, were used as the model system in this study. Chorionic tissue samples were obtained from CHS and control cat fetuses and cultures of cells were established. Acid phosphatase was utilized as a marker of lysosomes and cultures of chorionic fibroblasts from CHS and control fetuses were stained histochemically for acid phosphatase. The diameter of the largest lysosomes in 150 cells of each fetus was determined. The mean (+/- SD) diameter (in microns) of the largest lysosomes of normal fetuses was 0.9 +/- 0.13 (range 0.5-7.0 microns), whereas the mean diameter of lysosomes in CHS chorionic cells was 3.9 +/- 0.65 microns (range 0.5-25 microns). These means were significantly different (P less than 0.0001). These data suggest that it should be possible to diagnose human CHS in the first trimester by chorionic villus sampling.

Peripheral blood lymphocytes from boxer dogs with a history of cutaneous mast cell tumors were cultured for fragile site expression. As in a control group of dogs, cells from these dogs expressed folate-sensitive autosomal and X chromosome fragile sites. Cells from boxer dogs with mast cell tumors expressed the same three common fragile sites on the X chromosome as cells from control dogs. Three folate-sensitive autosomal fragile sites not observed in cells from the control dogs were identified in cells from boxers with mast cell tumors. These included fragile sites near the telomeres of the arms of chromosomes 3 and 4 and a fragile site on the distal half of chromosome 15. Cells from boxers with mast cell tumors showed a greater frequency of fragile site expression than did cells from control dogs, but this observation was attributed to an unintended selection bias for younger boxer dogs without mast cell tumors and older boxer dogs with mast cell tumors and an increased frequency of fragile site expression with increasing age in dogs of the boxer breed.

Peripheral blood lymphocytes from clinically normal Doberman pinscher and boxer dogs were cultured for folate-sensitive and, in preliminary studies, aphidicolin-inducible fragile site expression. Both autosomal and X chromosomal fragile sites were observed in canine cells cultured under folate/thymidine depletion and in cells cultured in medium containing aphidicolin. Results from the three dogs evaluated for both folate-sensitive and aphidicolin-inducible fragile site expression showed that the frequency of fragile site expression was significantly (P less than 0.05) greater in cells cultured in medium containing aphidicolin than in cells cultured in folate/thymidine-depleted medium. Cells from the boxer dog expressed a high percentage (66.67%) of aphidicolin-inducible fragile sites in contrast to the Doberman pinscher dog in which only 21.10% of the lymphocytes expressed aphidicolin-inducible fragile sites. The frequencies of spontaneous and folate-sensitive fragile site expression did not vary significantly by breed of dog. Age of dog was significantly and positively correlated with frequency of folate-sensitive fragile site expression in dogs of the boxer breed, but not in dogs of the Doberman pinscher breed. The dog X chromosome expressed three folate-sensitive and aphidicolin-inducible fragile sites. The G-band location of these three fragile sites showed homology with three recognized constitutive common fragile sites on the human X chromosome: Xp22, Xq21, and Xq27.2. Two specific autosomal fragile sites were identified, one on the distal end of the long arm of chromosome 1 and one on the distal end of the long arm of chromosome 8. Other autosomal fragile sites were also apparent but could not be assigned reliably to specific chromosomes.

Lysozyme from normal and genetically lysozyme-deficient rabbits was extracted from three types of tissue: leukocytic (bone marrow), lymphoepithelial (thymus and appendix), and gastrointestinal (colon). Extracts were analyzed by electrophoretic, chromatographic, and kinetic techniques. Identification of at least two isozymes of rabbit lysozyme was made with these techniques. The distribution of the isozymes was tissue specific. Leukocytic and gastrointestinal isozymes were clearly distinguished, and a possible lymphoepithelial isozyme that resembled the gastrointestinal isozyme electrophoretically and chromatographically but not kinetically was identified. Mutant, lysozyme-deficient rabbits lacked completely a detectable leukocytic isozyme but had gastrointestinal and lymphoepithelial isozyme(s) indistinguishable from those of normal rabbits. By electrophoretic methods, the mutant rabbits were demonstrated to lack a protein band corresponding to that of the leukocytic isozyme of lysozyme from normal rabbits. These observations considered collectively were interpreted as evidence that at least two primary isozymes of lysozyme are present in rabbits and that inherited lysozyme deficiency in rabbits is caused by the absence of a single primary isozyme.

The 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.

Chronic pneumonia was investigated in a litter of young Chinese Shar Pei in which 4 of 6 dogs were affected. Serum immunoglobulin concentrations (IgA, IgG, IgM) determined by radial immunodiffusion varied over time, but were not consistently lower in affected dogs, compared with control dogs. Two dogs that died had hydrocephalus and lymphoid depletion, in addition to severe broncho-pneumonia. Evaluation of ciliary ultrastructure in 2 affected dogs revealed random orientation of adjacent respiratory tract or oviductal cilia and a greater number of microtubular disarrangements, compared with control dogs. In vivo tracheal mucociliary clearance of 99mtechnetium macroaggregated albumin was absent in 1 dog examined. The ciliary abnormalities were suspected to have resulted in an inefficient mucociliary transport system predisposing to the development of pneumonia. Further evaluation of 1 Chinese Shar Pei revealed lymphocyte mitogenesis results that were not consistently less than those of a control dog, normal total hemolytic complement values, and normal blood neutrophil chemotaxis.

The 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.

Long term, low dose administration of adriamycin (ADR) to young growing rabbits resulted in significant alterations in bone structure and chemistry. Morphologic changes were most pronounced at epiphyseal and metaphyseal areas of long bones. Epiphyseal cartilage plates were thin and there was derangement of growth zones. Areas of primary and secondary spongiosa were deficient in trabeculae, osteoblasts and osteoclasts. Analysis of femora, humeri and lumbar vertebrae from ADR-treated rabbits revealed increased water and fat content and significant decreases in bone density compared to age-matched controls. Cortices of long bones were roentgenographically thin and contained large irregular spaces evident microscopically. Evaluation of bone ash from ADR treated rabbits revealed significant increases in the percentage of calcium and phosphorus, although Ca/P ratios were not different from controls. Results of in vitro studies indicate that ADR binds readily to nondemineralized, but not demineralized, fresh cortical bone powder. The findings of decreased bone density, histopathologic alterations, and a paucity of osteogenic cells in ADR treated rabbits are interpreted as retardation of bone maturation. It is suggested that ADR affects adversely both the organic and inorganic fractions of bone. Due to its unique characteristics of cytostatic action, binding to metal cations and orange-red fluorescence, ADR is a novel chemical agent that may be useful in experimental bone studies.

The existence of high-affinity [3H]-imipramine recognition sites was demonstrated in membranes prepared from the cerebral cortex, hypothalamus and platelets obtained from fawn-hooded rats. The Bmax and Kd values for [3H]-imipramine binding to cerebral cortical membranes were virtually identical to those obtained with cortical membrane preparations of Sprague-Dawley rats. An NBR strain of rats, genetically related to fawn-hooded rats, was found to have significantly higher levels of [3H]-imipramine binding sites in cerebral cortical membranes when compared to fawn-hooded and Sprague-Dawley rats. All four strains of rats examined possessed extremely high densities of [3H]-imipramine binding sites in a purified platelet membrane fraction. These results do not support the finding of others that the cerebral cortex and platelets of fawn-hooded rats are virtually devoid of [3H]-imipramine binding sites.

Twelve cats, all from research or commercial breeding colonies, had unidentified, intracellular organisms in the gastric mucosa. Histochemical staining and ultrastructural features provided the basis for identification of the organism as a Chlamydia sp. Ultrastructural observations were restricted to one of the 12 infected cats. There was no consistent association of gastric chlamydial infection and clinical disease. The infection was present in apparently healthy cats as well as those with a variety of clinical signs and lesions, especially weight loss of undetermined origin. None of the cats with gastric chlamydial infection had lesions compatible with feline pneumonitis. The significance of gastric chlamydial infection has yet to be established.

The serum susceptibility of 64 isolates of Haemophilus somnus from cattle was determined in a bactericidal assay with undiluted fresh or inactivated bovine serum with serial dilutions of bacterial suspension in RPMI 1640 medium. A total of 27 strains isolated from cattle with clinical disease (4 with thromboembolic meningoencephalitis, 13 with pneumonia, and 10 with reproductive failure) were compared with 35 strains from asymptomatic carriers (11 from the vagina and 24 from the prepuce). Essentially, all clinical isolates were serum resistant, whereas approximately 25% of preputial isolates were serum susceptible, as judged after 1 h of incubation in serum; a majority of vaginal isolates showed delayed serum susceptibility. Lysozyme played no role in serum killing, and the alternative complement pathway played only a minor role. Iron saturation, however, appeared to impart greater serum resistance to serum-susceptible strains from the vagina and prepuce. Perhaps the serum-susceptible strains from carriers would be useful vaccine candidates, but resistant strains from carriers may be pathogenic.

Laboratory studies were performed on 3 Collies with familial canine dermatomyositis, 6 progeny from a breeding of 2 of the Collies (incross litter), and 4 progeny from the breeding of an affected Collie male and a normal Labrador Retriever (outcross litter). Hematologic abnormalities were leukogram changes consistent with inflammation, and, in 2 severely affected incross dogs, anemia of inflammatory disease. Serum muscle enzyme activities were not markedly increased. High concentrations of immune complexes and proportionally increased total immunoglobulin G were present in the sera of moderately and severely affected incross dogs. The same dogs had weakly positive direct Coombs' tests, and 1 affected outcross dog had a strongly positive direct Coombs' test. Rheumatoid factor was present in a severely affected incross dog. A few dogs had polyclonal globulin increases that were attributed to inflammation. Low antibody titers to canine calicivirus were present in 3 dogs and a low titer to canine coronavirus was present in 1 dog.

Three Collies with a skin disorder, 6 progeny from a breeding of 2 of the Collies (incross litter), and the 4 progeny from the breeding of an affected Collie male and a normal Labrador Retriever female (outcross litter) were examined. By 7 to 11 weeks of age, all 6 dogs in the incross litter developed a qualitatively similar, but variably severe, dermatitis of the ears, face, lips, tip of the tail, and over bony prominences of limbs. Later, myopathic signs characterized by bilaterally symmetrical skeletal muscle atrophy of the head, neck, trunk, and extremities; facial palsy; decreased jaw tone; stiff gait; and hyperreflexia were observed in the dogs more severely affected by the dermatitis. Of the 4 dogs in the outcross litter, 3 had similar, but milder, clinical manifestations of the dermatitis and myopathy. Cutaneous lesions consisted of intraepidermal and subepidermal vesicles or pustules with intradermal infiltration by leukocytes. Muscle lesions included myositis; myofiber degeneration, regeneration, and atrophy; and fibrosis. A generalized myopathy in the severely affected dogs was indicated by abnormal readings on needle electromyograms and normal motor nerve conduction velocities. Spontaneous needle electromyogram abnormalities were fibrillation potentials, positive sharp waves, and bizarre high-frequency discharges. Retrospective and prospective genetic analyses disclosed a definite familial tendency and indicated the condition has an autosomal dominant component.

Studies were conducted to determine whether the gastric chlamydiae that have been observed recently in cats are of pathologic significance. Chlamydiae were isolated in mouse L cell cultures from the homogenized pooled gastric mucosa of 3 cats that had been identified, by histopathologic examination, to have gastric chlamydiosis. Ten specific-pathogen-free kittens were exposed by aerosol and oral inoculation to the harvested feline gastric chlamydiae cell-culture media. In general, the clinical signs and lesions were conjunctivitis, rhinitis, and mild gastritis. The clinical signs and lesions were most severe in 2 chlamydia-infected kittens that had received methylprednisolone acetate (50 mg/kg of body weight). Chlamydiae were demonstrated in epithelial cells of conjunctival and nasal smears in 10 of 10 infected kittens from postexposure days 7 through 35. In addition, chlamydiae were isolated in L cell cultures from a variety of antemortem and postmortem specimens from infected kittens. The present study provided evidence that feline gastric chlamydiae, under appropriate conditions, were capable of inducing, in cats, clinical signs and lesions similar to those induced by the feline pneumonitis agent.

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.

This study revealed that the lesions in platelets from cattle with the Chediak-Higashi syndrome (CHS) are not morphologically analogousto those present in the other granule-containing cells in cattle and other species with CHS. There was no evidence of enlargement of the lysosomal granules in bovine CHS platelets...

Rabbits receiving intramuscular injections of VX-2 carcinoma cells in biceps femoris muscles developed rapidly progressive neoplastic growths at 14 to 21 days associated with a significant hypercalcemia. The biologic behavior of the VX-2 carcinoma was characterized by local infiltration and metastases to regional lymph nodes and lungs. No metastases to skeletal tissues were evident. Femora from intramuscularly injected rabbits had varying degrees of osteophytosis and lysis evident roentgenographically. Histopathologic evaluation of femoral sections revealed periosteal new bone growth, cortical osteolysis, endosteal new bone growth, and in a few long term rabbits, pathologic fractures. Bone lesions were evident histologically in the vicinity of neoplastic growth (i.e., femora, tibiae) but not at distant sites (i.e., humeri and vertebrae). Mineral analyses of VX-2 carcinoma tissues and kidneys from VX-2-bearing rabbits revealed concentrations of calcium 83 and 3 times greater, respectively, than those of skeletal muscle and kidneys from controls. These findings correlated well with histochemical evidence of excessive amounts of calcium in sections of kidneys and VX-2 carcinoma tissues. Rabbits receiving intraperitoneal injections of VX-2 carcinoma cells did not develop hypercalcemia despite an extensive, progressive neoplastic burden with metastases to abdominal and thoracic viscera. Roentgenographic, histopathologic, and physiochemical analyses of selected bones from these rabbits revealed no significant alterations. These findings indicate that VX-2 carcinoma cells need to be in close proximity to skeletal tissues in order to induce hypercalcemia. The development of a significant hypercalcemia in intramuscularly injected rabbits precedes the invasion of osseous tissues by VX-2 carcinoma cells. Therefore, it appears that VX-2 carcinoma cells have the ability to alter skeletal morphology and physiochemistry through a dual humoral/cellular mechanism. The clinicopathologic characteristics of the VX-2 carcinoma in the rabbit suggest that the neoplasm is a good experimental model to study osseous-mediated hypercalcemia of malignancy.

Histologically, the mass had a variety of patterns. Some areas were composed largely of poorly organized matrix. It could not be determined whether the matrix was dentin, cementum, or osteoid, but other parts of the tumor had a matrix similar to dentin. In the more cellular areas of the tumor, long intrabranching epithelial strands were seen. The cells in the center of these strands spread apart, suggesting a stellate reticulum of the dental lamina. Cells near the basement membrane were elongated and seemed to stimulate surrounding stromal cells to produce a dentin-like matrix (fig. 2, 3). Occasionally an enamel-like matrix deposited by the epithelial cells was on top of the dentin matrix (fig. 4). The apparent inductive interassociation of the epithelial cells and the stromal cells to form dentin and enamel matrix resembled the process of odontogenesis. The stroma throughout the mass was similar to dental pulp and consisted of a highly cellular population of small mesenchymal cells with a clear background and scant collagen deposition. This mass caused no apparent clinical disease in the sheep and had not been noticed until necropsy. The radiographic appearance of smooth, distinct boundaries suggests that the mass was a slow-growing tumor. Similar tumors occur in man, but are rare. They are slow-growing and locally destructive but they do not metastasize [4].

A ten year old Hereford bull with Chediak-Higashi syndrome was examined at necropsy after a lifelong history of recurrent bacterial infections. Amyloidosis, which has not been previously reported in Chediak-Higashi, was identified in liver, spleen and kidney.

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.

A genetic disorder of rabbits consisting of a deficiency of the enzyme lysozyme is characterized. The condition appears to be inherited as an autosomal recessive trait. Most of the tissues of lysozyme-deficient rabbits including bone marrow, liver, lung. spleen and bone had levels of lysozyme which were 1% or less of the levels in the corresponding tissues of normal rabbits when measured with the lysoplate method. Levels of lysozyme in the kidney and serum were 6% of controls, but the thymus of the lysozyme-deficient rabbits had normal levels of the enzyme. All leukocytes of the lysozyme-deficient rabbits were negative for lysozyme when examined by a histobacterial technic. No morphologic lesions could be detected in any of the tissues of the lysozyme-deficient rabbits. Although several species of animals have been reported to be lysozyme deficient, this appears to be the first report of lysozyme deficiency occurring as a mutant condition. It is suggested that these mutant rabbits may be useful as a resource for experiments designed to delineate the biologic role of lysozyme.

In this study, the serum sensitivity of 23 P. haemolytica isolates and 18 P. multocida isolates was determined by incubating dilutions of bacteria with equal volumes of fresh or heat-inactivated bovine serum for one, two, or three hours. Clinical isolates of both Pasteurella species were resistant to serum, whereas isolates from asymptomatic cattle varied in serum susceptibility. The classical pathway of complement appeared to be the principal means of complement mediated killing as detected by incubation in the presence or absence of EGTA-MgCl2. Lyzozyme and iron saturation of serum did not greatly affect serum susceptibility with either of the Pasteurella species.

Ocular examinations were performed on cattle, cats, mink, and mice affected with Chédiak-Higashi syndrome (CHS). Bovine eyes were examined grossly and with an indirect ophthalmoscope, and Schirmer tear tests were performed. Feline eyes were examined grossly as well as with an indirect opthalmoscope and a slit lamp biomicroscope, and Schirmer tear tests were done on them. Postrotatory nystagmus was induced and measured in clinically normal Siamese cats, in clinically normal Persian and domestic short-haired cats, and in cats with CHS. Mink and mouse eyes were examined grossly with focal illumination. The animals with CHS had photophobia, pale irises, and fundic hypopigmentation associated with red fundic light reflections. Cats with CHS also had cataracts. Spontaneous nystagmus was observed in four of nine cats with CHS, and the duration of induced nystagmus was longer in the cats with CHS and in Siamese cats than in clinically normal cats that were not Siamese. Tear secretion appeared to be normal in all species of animals with CHS. The ocular manifestations of CHS in these animals were compared with those reported in man and were found to be similar.

Lysozyme activities in gastrointestinal and lymphoreticular tissues and in macrophage culture products of normal and genetically lysozyme-deficient rabbits were measured and compared. The supernatants of homogenates of 18 gastrointestinal and lymphoreticular tissues were assayed for lysozyme. In the gastrointestinal tract, there were no major differences in lysozyme activities between the two groups of rabbits. Ascending colon and orad gastrointestinal tissues had low activities, whereas descending colon and rectum had very high lysozyme activities in both types of rabbits. In the lymphoreticular tissues, lysozyme-deficient rabbits had graded lysozyme deficiencies: slight or no deficiency was present in thymus, partial deficiency in gut-associated lymphoid tissues, and near total deficiencies in secondary lymphoid tissues and bone marrow. Both macrophage culture cells and fluids from lysozyme-deficient rabbits were completely deficient in lysozyme. The results indicate that in the lysozyme-deficient rabbits there is tissue-specific lysozyme activity expressed and when considered along with other observations suggest that the lysozyme-deficient condition may be due to a deficiency of an isozyme or isozymes of lysozyme.

The genetic aspects of inherited, insulin-dependent diabetes mellitus of Keeshond dogs were studied retrospectively and in a prospective mating program. The symbol dm was used to designate the gene that causes hypoplasia of the islets of Langerhans. The retrospective study disclosed 4 diabetic dogs; prospective outcross, backcross, and inbred matings disclosed 49 diabetic dogs. Outcrossing demonstrated that the diabetic phenotype was displayed readily against a genetic background of a breed other than the Keeshond. In dogs with the dm/dm genotype, onset of diabetes was most frequent before the dog was 6 months old, but did occur in some older dogs. The dm genotype was best described as autosomal recessive.

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.

Ollulanus tricuspis were found in the stomachs of 26 of 201 cats which were mainly from Washington and Idaho. Twenty-four of the cats were from research or commercial breeding colonies (catteries) and two were pets. Twenty-seven percent of colony cats were infected with O. tricuspis, whereas only 2% of the pet cats were infected. No consistent clinical signs were seen. Histologically there was a significant increase in fibrous connective tissue in the lamina propria and in the number of lymphoid follicles and globule leukocytes in the gastric mucosa of infected cats. Histologic examination revealed no parasites in 13 of the 26 infected cats. The 26 infected cats were identified by examination of stomach washings. An additional 21 cats had larvae of other nematodes in their stomachs. These nematode larvae need to be differentiated from O. tricuspis.

Familial canine dermatomyositis in collie dogs is a newly recognized spontaneous disease that resembles dermatomyositis in children. A litter of 9 collie dogs was studied from birth to 7.5 months of age. The onset and severity of dermatitis and myositis correlated with elevated serum levels of circulating immune complexes (CICs) and IgG. The immunoglobulin component of the CICs consisted principally of IgG. All dogs developed elevated levels of CIC before or concurrent with the onset of dermatitis. Myositis developed later. The CIC tended to peak between 14-18 weeks of age in all dogs except the most severely affected dog, in which the CICs continued to increase to 238 micrograms/ml (controls 30 micrograms/ml) at 7 months of age. In the moderately affected dogs the CICs tended to stabilize at the levels reached at 14-18 weeks, and in the mildly affected dogs the CICs tended to decrease to normal levels after 14-18 weeks. Although the dogs had electromyographic and repetitive nerve stimulation abnormalities, the abnormalities did not correlate with severity of dermatomyositis or degree of elevation of CICs or IgG. Necropsy at 7.5 months of age revealed that all dogs had myositis and 8 of 9 had dermatitis. Except for 1 dog, the severity of dermatomyositis correlated positively with higher levels of CICs. A strong positive association between elevated levels of CICs and IgG, but not IgM or IgA, was generally present. Elevated levels of CICs appear to be involved in the mechanisms that control the development, severity, and progression of dermatomyositis in collie dogs.

Initial clinical, genetic, cytochemical and ultrastructural studies have characterized the Chediak-Higashi syndrome in cats. Three cats with Chediak-Higashi syndrome were found in a single line of 27 Persian cats, and three additional affected cats were produced from two prospective breedings of the original line. The disorder was characterized genetically as an autosomal recessive condition. All cats in the line with the combination of yellow eye color and "blue smoke" hair color exhibited the disorder. Four of the five cats examined had bilateral nuclear cataracts as early in life as 3 months of age. No increased susceptibility to infectious disease was observed. A bleeding tendency was noted. Abnormally large eosinophilic, sudanophilic, peroxidase-containing granules were observed in the neutrophils of the granulocytic series of blood and bone marrow by electron and light microscopy. Granules of eosinophils and basophils were also enlarged. Light microscopic studies of hair and skin revealed enlarged melanin granules. These manifestations were similar to those in man, mink, cattle, mice, and the killer whale with Chediak-Higashi syndrome. Cats are the sixth species in which this genetic disease has been reported.

The Chediak-Higashi syndrome (CHS) occurs in mammals, including humans and cats. The CHS is characterized by decreased oculocutaneous pigmentation, enlarged cytoplasmic granules, increased susceptibility to infections, and a hemorrhagic tendency. Ocular anomalies include pale irides and albinotic or subalbinotic fundi. Cats with CHS also have photophobia and prolonged postrotatory nystagmus. Since hypopigmentation of the pigment epithelium is correlated with misrouting of retinal ganglion cells in mammals, visual projections of CHS cats were examined by autoradiographic techniques to determine whether they exhibit abnormal retinogeniculate projections. In CHS cats, misrouted optic projections fragment layer A1 of the dorsal lateral geniculate nucleus into several islands, similar to the disruption of this lamina reported in the Siamese cat.

The inheritance of coat color, hooded-coat pattern, and platelet storage pool deficiency of fawn-hooded rats was studied by crossing fawn-hooded rats with rats of other strains. It was determined that the tan coat color and the platelet storage pool deficiency were pleiotropic effects of the autosomal recessive red-eyed dilution (r) gene. The hooded-coat pattern was determined to be the effect of a different autosomal recessive gene. It also was demonstrated that the tan coat color of fawn-hooded rats is a dilution and that the shade of the tan coat color expressed varied with the underlying coat color genes.

The eyes of 7, 9, and 11-year-old Chediak-Higashi syndrome (CHS)-affected cats were examined by light, fluorescence, and electron microscopy. Numerous round to oval bodies of various sizes were associated with the retinal pigment epithelium (RPE). These bodies stained positively with periodic acid-Schiff. They also displayed a bright yellow autofluorescence and stained positively with a prolonged Ziehl-Neelsen acid-fast method for demonstration of lipofuscin, suggesting that they contained lipofuscin or a lipofuscin-like material. Ultrastructural examination disclosed the bodies to be secondary lysosomes and large to giant-sized residual bodies. Many of the residual bodies were extracellular and formed drusen-like mounds, covered by deposits of basal lamina, beneath the RPE. Also evident were scattered degenerated RPE cells and other RPE cells that had detached and migrated into the interphotoreceptor space. The presence of drusenoid bodies, and the loss of cells from the RPE monolayer in CHS eyes have not been reported previously. Many of the changes in the CHS cat eyes resemble those in non-CHS aging eyes of man and other species.

Muscles from male and female C57BW6J Chediak-Higashi syndrome (CHS) and phenotypically normal mice with the bgJ allele were studied microscopically and histochemically for the presence of basophilic cytoplasmic structures seen by other investigators in muscles of CHS mice of the SB/k strain. Triceps brachii, gastrocnemius, quadriceps femoris, and biceps femoris muscles were examined. Multiple basophilic cylindrical lesions were present in hematoxylin and eosin-stained muscle from all groups. Lesions were positive for esterase, Sudan black, and periodic acid-SchiK Lesions were only seen in type I1 muscle fibers. Type I muscle cells comprised less than an estimated 5% of the total muscle fibers in the four muscles examined. Scores were assigned based on the presence or absence of lesions in each muscle. Male mice of both phenotypes had significantly more lesions (P 0.05) between lesions of pheno- typically normal female and female CHS mice. Basophilic cytoplasmic structures did not prove to be a mani- festation of the CHS trait. Chediak-Higashi syndrome (CHS) is an inherited disease characterized by excessively large cytoplasmic granules, oculocutaneous albinism, photophobia, a bleeding tendency, and reduced resistance to infec- tion.17 Enlarged granules occur in many cell types in- cluding those of the pancreas, pituitary, liver, adrenal cortex, spleen, kidney, scent gland, bone marrow, hair, skin, iris, and gastric and duodenal mus~osa.'~J~ The disorder is inherited as an autosomal recessive trait, and the trait in mice has been designated Muscle lesions have been reported in CHS mice of the SB/Le strain.yJ2 These lesions consist of single or multiple cylindrical structures within the cytoplasm devoid of myofibrils, are basophilic when stained with hematoxylin and eosin (HE), and are positive with reactions for periodic acid-Schiff (PAS), non-specific esterase, l2 and Sudan black.I0 Negatively reactive areas are present when stained for myofibrillar adenosine triphosphatase (ATPase) and the oxidative enzyme succinic dehydrogenase (SDH).y Electron microscopic studies disclosed that these core-like lesions consisted of either membranous structures in parallel-tube or honey-comb configurations or amorphous, flu@ ma- terial with no recognizable cellular organelles present in the core-like structures." There were no reports of similar lesions in any of the other species with CHS. Preliminary studies with C5 7BL/6 J beige mice were unable to confirm the observations made with SB/Le beige mice. The present study was designed to deter- mine whether skeletal muscle lesions of the type de- scribed above are present in C57BL16J beige mice.

Ollulanus tricuspis, a minute gastric nematode not previously documented in the United States, was identified by histologic examination and by dissecting microscopic examination of stomach contents of 7 cats from 3 breeding colonies in the Pacific Northwest. The nematode may have contributed to chronic weight loss and undernutrition in part by causing gastric irritation, with resultant vomiting. The findings suggest that infection with Ollulanus parasites may be more widespread in the United States than realized.

A spontaneous apparently unique auricular chondritis in the pinna of fawn-hooded rats is described. The chondritis was bilateral, with adult onset, and resulted in a marked thickening of the auricular cartilage. Microscopically, islands of proliferative cartilage were present, and at the margins between the normal cartilage and the thickened abnormal cartilage a marked cellular inflammatory response was present. The condition was familial in rats of the fawn-hooded strain and appeared to be unrelated to the platelet storage pool deficiency in this strain of rats. Biochemically no increased synthesis of pinna cartilage was detected. No histopathologic lesions were detected in other cartilaginous tissues of affected rats. The lesions in the pinna bore a striking resemblance to those induced in rats by immunization with Type II collagen. The spontaneous condition described herein may, therefore, represent a unique model of relapsing polychondritis of human beings, a disease with auricular chondritis associated with antibodies to Type II collagen.

The ultrastructural of platelets from Chédiak-Higashi (CH) and normal cattle, mink, and cats at rest was studied. Platelets from CH animals had a virtual absence of platelet dense granules. Alpha granules, amorphous membrane-surrounded structures, mitochondria, and microtubules of CH bovine platelets were similar in number and appearance to those in normal bovine platelets. Giant CH granules, present in other cells and considered diagnostic of the syndrome, could not be identified in platelets from CH animals. The open canalicular system and dense tubule system were not readily identifiable in resting bovine platelets. The ultrastructure of normal and CH cattle platelets was evaluated at various stages of ADP-induced aggregation. After platelets changed shape during the first phase of aggregation, the ultrastructural appearance of CH platelets was similar to that of normal platelets. The CH platelets composing the aggregates during irreversible aggregation did not appear as activated as did normal platelets, even though the aggregation tracings were similar. Normal and CH cattle platelets treated with thrombin appeared morphologically similar and were characterized by centrifugal movement of granules.

The Maltese dilution is an autosomal recessive trait of cats that dilutes black cats to blue, and orange cats to cream. The pigmented cutaneous and ocular tissues of Maltese dilution and control cats were examined and compared by light microscopy. Most of the melanin granules in all of the pigmented cutaneous tissues of the Maltese dilution cats were aggregated together into large clumps. However, none of the intraocular tissues containing melanin producing cells of either neural crest or optic cup embryologic origin contained clumped melanin granules. It is concluded that the Maltese dilution trait is a unique generalized albinism without ocular involvement.