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Type II tyrosinemia, designated Richner-Hanhart syndrome in humans, is a hereditary metabolic disorder with autosomal recessive inheritance characterized by a deficiency of tyrosine aminotransferase activity. Mutations occur in the human tyrosine aminotransferase gene, resulting in high levels of tyrosine and disease. Type II tyrosinemia occurs in mink, and our hypothesis was that it would also be associated with mutation(s) in the tyrosine aminotransferase gene. Therefore, the transcribed cDNA and the genomic tyrosine aminotransferase gene were sequenced from normal and affected mink. The gene extended over 11.9 kb and had 12 exons coding for a predicted 454-amino-acid protein with 93% homology with human tyrosine aminotransferase. FISH analysis mapped the gene to chromosome 8 using the Mandahl and Fredga (1975) nomenclature and chromosome 5 using the Christensen et al. (1996) nomenclature. The hypothesis was rejected because sequence analysis disclosed no mutations in either cDNA or introns that were associated with affected mink. This suggests that an unlinked gene regulatory mutation may be the cause of tyrosinemia in mink.

Control of a naturally occurring lentivirus, equine infectious anemia virus (EIAV), occurs in most infected horses and involves MHC class I-restricted, virus-specific CTL. Two minimal 12-aa epitopes, Env-RW12 and Gag-GW12, were evaluated for presentation by target cells from horses with an equine lymphocyte Ag-A1 (ELA-A1) haplotype. Fifteen of 15 presented Env-RW12 to CTL, whereas 11 of 15 presented Gag-GW12. To determine whether these epitopes were presented by different molecules, MHC class I genes were identified in cDNA clones from Arabian horse A2152, which presented both epitopes. This horse was selected because it is heterozygous for the SCID trait and is used to breed heterozygous females. Offspring with SCID are used as recipients for CTL adoptive transfer, and normal offspring are used for CTL induction. Four classical and three putative nonclassical full-length MHC class I genes were found. Human 721.221 cells transduced with retroviral vectors expressing each gene had equine MHC class I on their surface. Following peptide pulsing, only cells expressing classical MHC class I molecule 7-6 presented Env-RW12 and Gag-GW12 to CTL. Unlabeled peptide inhibition of 125I-labeled Env-RW12 binding to 7-6-transduced cells demonstrated that Env-RW12 affinity was 15-fold higher than Gag-GW12 affinity. Inhibition with truncated Env-RW12 demonstrated that amino acid positions 1 and 12 were necessary for binding, and single substitutions identified positions 2 and 3 as possible primary anchor residues. Since MHC class I 7-6 presented both epitopes, outbred horses with this allele can be immunized with these epitopes to optimize CTL responses and evaluate their effectiveness against lentiviral challenge.

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.

The autosomal recessive disease Chediak-Higashi syndrome (CHS) is a progressive and generally fatal disease of humans. The under lying 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 μm) of the largest lysosomes of normal fetuses was 0.9 ± 0.13 (range 0.5–7.0 μm), whereas the mean diameter of lysosomes in CHS chorionic cells was 3.9 ± 0.65 μm (range 0.5–25 μm). These means were significantly different (P < 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 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.

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.

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.

Chediak-Higashi syndrome (CHS) is an autosomal recessive disease in humans, cats, and 8 other species. The homology of CHS in humans and cats has been demonstrated. Since human CHS is a progressive, serious, and eventually fatal disease, a method for prenatal diagnosis would be desirable. This study was designed to determine whether CHS could be diagnosed prenatally by examination of amniotic fluid cells. The amniotic fluid samples were obtained from CHS and control cat fetuses on the 45th day of gestation and cultures of cells were established. Because the underlying enzyme deficiency in CHS has not been identified, it was necessary to use a secondary manifestation of the syndrome in these studies. The secondary manifestation used was the characteristic enlargement of lysosomes associated with the disease. The lysosomes of these cells were stained by acid phosphatase histochemistry and the diameter of the largest lysosome in each cell was measured by light microscopy with a calibrated ocular micrometer. The diameters of the largest lysosomes in cells of normal fetuses ranged from 0.5 to 7.0 micron (means ranged from 0.9 to 1.8 micron), whereas the diameter of the largest lysosomes in the cells of CHS fetuses ranged from 0.5 to 30 microns (means ranged from 6.4 to 12.8 microns). The approximate t-test for independent samples with unequal variances disclosed that the largest acid phosphatase-positive lysosomes in amniotic fluid cells of CHS cat fetuses were significantly larger than the lysosomes in the cells of normal cat fetuses (P less than 0.0001). This information should, by extrapolation, provide the basis for the prenatal diagnosis of human CHS by amniocentesis.

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.

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.

Auditory brainstem responses ABRs were recorded in cats with Chediak-Higashi syndrome using monaural stimulation. The components appearing between 1 and 3 ms after stimulus onset were greatly attenuated in the ABRs recorded using a reference contralateral to the stimulated ear. These data suggest that abnormalities exist in the brainstem auditory pathway in the region of the superior olivary complex in cats with Chediak-Higashi syndrome.

The distribution of the enzyme tyrosine aminotransferase in tissues of mink, Mustela vison, was investigated. High levels of enzymatic activity were detected only in liver, documenting the hepatic-specific nature of this enzyme in this species. Further studies disclosed that tyrosine aminotransferase is not absent from non-hepatic tissues because of the lack of the use of a stabilized buffer, sensitivity to temperature, or due to the presence of an inhibitor. Collectively, these results suggest that the enzymatic assay of tyrosine aminotransferase will be unlikely to be an efficacious approach for identifying mink that are heterozygous for the autosomal recessive deficiency of this enzyme that is common in dark mink.

Nonspecific cytotoxic cell (NCC) activity was assessed in the peripheral blood of four isogenic lines of rainbow trout (Oncorhynchus mykiss ) which were derived by the chromosome set manipulation technique of androgenesis. In these fish, whose isogenicity was previously confirmed by multilocus DNA fingerprint analysis, NCC activity was studied by the release of 51 Cr from YAC-1 targets. Two groups of trout (the homozygous Arlee 12 line and the heterozygous hybrid of the Arlee 63 and Arlee 12 lines) had significantly lower levels of NCC activity in peripheral blood than either outbred rainbow trout or other lines with Hot Creek or hybrid Arlee × Hot Creek ancestry. The low NCC activity in the Arlee line appears to be inherited as a recessive trait. Peripheral blood cells of the trout mediated lectin dependent cellular cytotoxicity (LDCC) with the addition of phytohemagglutirain to co-cultures of effector cells and YAC-1 cells. The low NCC activity in the peripheral blood of these fish is not due to a condition analogous to the NCC-deficient Chediak-Higashi syndrome of man or the beige mutation of mice.

Cytogenetic investigations of the domestic dog, Canis familiaris, were performed on one Doberman pinscher and two Boxer dogs. Conventional homogeneously stained and G-banded metaphases from peripheral blood lymphocyte cultures synchronized with amethopterin and bromodeoxyuridine were studied. These procedures permitted the unequivocal identification of all canine chromosomes. A canine chromosome idiogram was constructed on the basis of the G-banding pattern at the haploid 327-band resolution level. The secondary constrictions and tapering of the telomeric regions characteristic of several canine chromosomes are described. Q-, C-, and NOR-banding were also performed and the salient features are described. This karyotype should enhance the value of the canine species in cytogenetic investigations.Key words: canine karyotype, G-banding, Q-banding, C-banding, NOR-banding, cell synchronization idiogram.

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.

Mice of the C57BL/6 strain with the Chediak-Higashi syndrome (CHS) have a dark gray (charcoal gray) coat color. No other abnormalities are apparent grossly.

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.

Genetically determined C3 deficiency in Brittany spaniel dogs shares a number of biochemical and clinical characteristics with the human disorder. In humans, the gene for C3 deficiency is a null gene that is allelic to the structural gene for C3 and is not linked to the major histocompatibility locus. The current study used allotype analysis of canine C3 in order to demonstrate that the gene for C3 deficiency in these dogs is also a null gene allelic to the structural gene for C3. In addition, preliminary pedigree analysis suggests that the gene for canine C3 deficiency is apparently not closely linked to the major histocompatibility complex of the dog. Thus, it appears that C3 deficiency in Brittany spaniel dogs not only shares biochemical and clinical features with C3 deficiency in humans, but also shares some genetic characteristics with the human disorder.

The phenotypes of 273 offspring of 56 matings of various genotypes of lysozyme-deficient and normal New Zealand white rabbits were analyzed. It was determined that lysozyme deficiency of rabbits is inherited as an autosomal recessive with complete penetrance. The symbol ld is suggested for the gene for lysozyme deficiency. Further studies on the nature of the condition revealed that the lysozyme deficiency was not due to a lysozyme that was cold labile, or to a lysozyme that was so tightly bound it was unavailable for enzymatic assay, or to a lysozyme with a variant pH optimum. It was demonstrated, however, that the pH profile of the lysozyme remaining in the ld/ld rabbits was different from that found in normal rabbits.

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.

Cats with the Chediak-Higashi (CH) syndrome have abnormal hemostasis with prolonged bleeding times and normal coagulation times. Platelet aggregation induced by serotonin, ADP, and collagen was impaired. Platelets from normal and CH cats were incubated with 14C-adenine and then gel-filtered. Gel-filtered platelets (GFP) from CH cats contained 63% of the ATP, 38% of the ADP, 100% of the Ca2+, and 75% of the Mg25 of normal platelets. Serotonin could not be detected in CH platelets. Acid hydrolase and total platelet protein of CH platelets was similar to normal platelets. Gel-filtered platelets were treated with thrombin to induce maximal secretion. Secretion of ATP, Ca2+, and Mg2+ was 1.9%, 12.4%, and 16% respectively of normal platelets. ADP secretion by CH platelets was not detectable. The ATP/ADP ratio in the 14C-labeled metabolic pool of normal platelets was similar to that of total measured nucleotide pool of CH platelets. These findings suggest that in feline CH platelets, as in platelets from CH mink and cattle, there is storage pool deficiency that is virtually complete, and the virtual absence of ADP and 5HT may in part account for the abnormal hemostasis. Aggregation of platelets from CH cats was impaired, but these platelets did aggregate to arachidonate, serotonin-induced biphasic aggregation, and the aggregation response to ADP and collagen varied according to the amount of serotonin-induced TxB2 formed. These findings support a major role for arachidonate in platelet activation.

Greig cephalopolysyndactyly syndrome is an autosomal dominant form of complex polydactyly in man. Attention is called to the evidence that, on both morphological and comparative gene mapping grounds, this defect is homologous to Xt-extra toes in the mouse. The pattern of polydactyly in both species is very similar. In addition, both conditions probably map close to the T-cell receptor gamma polypeptide at 13 A2-3 in mouse and 7p15 in humans.

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.

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.

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.

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.

CH50, C4, C2, and C3 levels were evaluated in 7 dogs affected with dermatomyositis and in 22 control dogs. Dogs with dermatomyositis did not have clinical evidence of active disease at the time of serum collection for complement assays. No absolute complement component deficiency was identified in dermatomyositis-affected dogs in this study; however, a statistical difference in C2 was identified between control dogs of non-collie breeds and control collies, suggesting there may be a breed difference in complement levels.

1. 1. The distribution of lysozyme in tissues and fluids of ruminants was examined and it was determined that, except for a few tissues, ruminants were deficient in lysozyme activity compared with other species. 2. 2. The prominent exception was the abomasum of cattle, which had high levels of lysozyme activity. 3. 3. Mixing and extraction studies indicated that the lysozyme deficiency of ruminants was due neither to the presence of inhibitors of lysozyme in ruminant tissue nor to the binding of lysozyme in a manner that interfered with its enzymatic activity or assay. 4. 4. Other investigations have indicated that isozymes of lysozyme are present in ruminants and this study suggests that ruminants have only low levels of the isozyme that is the major isozyme of lysozyme in non-ruminants.

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.

The Chediak-Higashi syndrome (CHS) is a hereditary disorder of man, with the homologous condition reported in five animal species. Multiple defects, including oculocutaneous hypopigmentation, are present in individuals with this syndrome. Giant cytoplasmic granules, including melanosomes and lysosomes, are characteristic. In this study, eyes from CHS affected and control cats, cattle, mink, and mice were examined histologically to determine: 1) degree of pigmentation; 2) structure and distribution of melanin granules; and 3) morphology of cells and tissues containing melanin. The CHS cattle were found to be the most ocularly hypopigmented species, whereas CHS mouse eyes contained considerably more melanin than those of the other species. Melanin granules of abnormal sizes and shapes were present in neuroepithelial and uveal tissues of CHS animals of all four species. Depigmentation apparently had occurred in the CHS eyes, since less melanin was present in eyes of old CHS animals of each species than was present in eyes of young animals. In addition, melanin containing macrophages were common in CHS eyes, and the numbers of melanocytes and pigmented epithelial cells were decreased in older CHS eyes.

Prolonged mean template bleeding time of 14 min observed in seven cattle with the Chediak-Higashi syndrome (CHS) prompted the examination of platelet function in these animals. There was no distinguishable difference in concentration of circulating platelets between CHS and control cattle. CHS bovine platelets failed to aggregate in vitro in response to concentrations of acid-soluble collagen which induced aggregation in all normal samples. The primary platelet response to 5 muM ADP was normal in CHS cattle. A markedly decreased amount of serotonin (1.2% of normal) was detected in CHS bovine platelets. Bovine CHS platelet ATP and ADP contents were significantly less than normal, and the ATP/ADP ratios were 5.04 in normal and 29.38 in CHS platelets. Results of these animal investigations prompted a similar study of two patients with CHS. In humans, an increased bleeding time greater than 15 min and an in vitro impaired aggregation response to acid-soluble collagen and 5 muM adrenaline were discovered. Both ATP and ADP were reduced in CHS human platelets, and the ATP/ADP ratio was 3.96, compared to a ratio of 1.52 for platelets of two normal subjects. These findings suggested the presence of a “storage pool disease” of CHS platelets.

The Chediak-Higashi syndrome is a genetic disorder characterized by greatly enlarged cytoplasmic granules, including lysosomes and melanosomes. Eyes of humans and animals with Chediak-Higashi syndrome are hypopigmented to various degrees. Intraocular melanin granules vary in size, with some being massively enlarged. Electron microscopie examination of retinal pigmented epithelium of kittens with Chediak-Higashi syndrome disclosed a number of abnormalities of premelanosomes and melanosomes. Few premelanosomes were present. Most of the melanin granules were giant sized, but their structures varied. Some of the giant granules were composed of several premelanosomes and melanosomes in different stages of maturation. Others contained randomly oriented melanofilaments between melanosomes. There were also complex giant granules consisting of both melanosomal and lysosomal components. Inappropriate fusion of cytoplasmic granules appears to be the most likely mechanism for formation of the giant granules. Fusion of premelanosomes with lysosomes and resultant destruction of the premelanosomes probably is a major cause of the ocular hypopigmentation of Chediak-Higashi syndrome.

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.

Aggregation of human platelets in vitro can occur in two phases—primary and secondary aggregation1. Primary aggregation is due to the direct interaction of the aggregating agent with its receptor2,3, whereas secondary aggregation is associated with both the secretion of dense granule constituents, including the aggregating agents, serotonin and ADP (ref. 4) and the activation of a pathway for the conversion of arachidonic acid to prostaglandins (PGs)5. Both secondary aggregation and the associated secretion of dense granule constituents are contingent on the formation of PGG2 and PGH2 which can, in part, be transformed into thromboxane (TX) A2 (refs 1,3,6,7). TXA2 is a potent aggregating agent and apparently the functionally active compound in the arachidonate pathway8–10. However, the precise role of TXA2 in mediating secondary aggregation is a matter of controversy as it is not clear whether secondary aggregation can occur independently of secretion. We report here that platelets from cats with the Chediak–Higashi (CH) syndrome11 aggregate with arachidonate in a cyclooxygenase-dependent manner and that biphasic aggregation produced by serotonin is accompanied by TXB2 formation without detectable ADP secretion. These results demonstrate that secondary platelet aggregation can be mediated through thromboxane formation without participation of secreted ADP.

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.

3 Bonniwell MA, Barlow RM: Ataxia/paresis syndrome of sheep in West Africa associated with bilateral multifocal cerebrospinal poliomalacia. Vet Rec 116:94, 1985 4 Cordy DR, East NE, Lowenstine LJ: Caprine encephalomalacia. Vet Pathol 21:269, 1984 5 Degirolami U, Zivin JA: Neuropathology of experimental spinal cord ischaemia in the rabbit. J Neuropath Exp Neurol 41:129, 1982 6 Innes JRM, Plowright W: Focal symmetrical poliomalacia of sheep in Kenya. J Neuropath Exp Neurol 14: 185, 1955 7 O’Sullivan B M, Blakemore W F Acute nicotinamide deficiency in the pig induced by 6-aminonicotinamide. Vet Pathol 17:748, 1980 8 Ten Eyck FW, Martin WJ, Kernohan JW: Acute porphyria; necropsy studies in nine cases. Mayo Clin Proc 36:409, 1961 9 Tschudy DP, Bonkowsky HL: Experimental porphyria. Fed Proc 31:147, 1972 10 Wilson TM, Drake TR: Focal symmetrical poliomyelomalacia. Can J Comp Med 46:218, 1982

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.

A male collie aged 5 years 10 months, which developed dermatomyositis at 2 months of age, died from severe secondary amyloidosis. Amyloid deposition was most severe in renal glomeruli and produced renal failure. Amyloidosis has been reported in man with immune-mediated disorders including rheumatoid arthritis, systemic lupus erythematosus and dermatomyositis. It is possible that the inflammation in this case of familial canine dermatomyositis may have predisposed to the development of amyloidosis.

Platelets from cats with the Chediak-Higashi syndrome (CHS) have an intrinsic platelet defect similar to storage pool deficiency in man and there is a virtual absence of secretable serotonin and ADP. Serotonin-induced platelet aggregation responses were monitored in four cats with the CHS. Low serotonin concentrations (0.5 ug/ml) induced a monophasic reversible aggregation response. When the concentration was increased (1-2 μg/mlj a biphasic aggregation response was observed and at higher concentrations (4-8 μg/ml) a monophasic irreversible aggregation wave was observed. The concentration dependent aggregation responses can be progressively inhibited with increasing concentrations of the serotonin antagonists, SQ-10,631 and cyproheptadine. The secondary aggregation wave is inhibited by both aspirin and indomethacin. Arachidonate (0.5mM) induces a monophasic irreversible CHS olatelet aggregation resoonse which is also inhibited aspirin and indomethacin. These findings suggests that the secondary aggregation wave is due to an arachidonate metabolite formed in response to serotoninWhile the metabolite has not been directly identified serotonin induces formation of thromboxane B2 (TxB2) by CHS cat platelets and in all but one animal increasing Serotonin increased TxB2 formation. In addition, the cat whose platelets exhibited the weakest serotonm-induced aggregation response produced the smallest amount of TxB2.