Classification of canine nonangiogenic, nonlymphogenic, gastrointestinal sarcomas based on microscopic, immunohistochemical, and molecular characteristics.

Canine nonangiogenic, nonlymphogenic, gastrointestinal sarcomas have been previously diagnosed as gastrointestinal stromal tumors (GIST), leiomyosarcomas, or nonspecified spindle cell sarcomas, but diagnostic criteria for each entity are poorly defined. We propose a classification for canine nonangiogenic, nonlymphogenic, gastrointestinal sarcomas based on microscopic, immunohistochemical, and molecular characteristics. Applying the classification to 40 canine nonangiogenic, nonlymphogenic, gastrointestinal sarcomas documented its diagnostic and prognostic value. Eighteen (45%) sarcomas were classified as GIST based on positive KIT immunoreactivity. All GISTs were positive for vimentin, 14 (78%) were positive for S-100, and 6 (33%) were positive for smooth muscle actin (SMA). In contrast to their human counterparts, canine GISTs occurred mainly in the small intestine (67%) but commonly metastasized (5/18) to liver, lymph nodes, and omentum. Six GISTs had an activated KIT mutation in exon 11 of c-Kit, but no mutations were detected in exons 8, 9, 13, and 17. Twelve (30%) sarcomas were classified as leiomyosarcomas based on positive labeling for SMA and negative labeling for KIT. Four of these neoplasms were well differentiated leiomyosarcomas characterized by weak to no labeling for vimentin, and 8 were poorly differentiated leiomyosarcomas characterized by strong labeling for vimentin. None of the leiomyosarcomas metastasized, but poorly differentiated leiomyosarcomas had a higher risk of local invasion. Ten (25%) sarcomas were classified as non-GIST/nonleiomyosarcomas that were negative for KIT and SMA but positive for vimentin and either S-100 and/or PGP 9.5. These neoplasms most likely represent sarcomas of neurogenic differentiation resembling Schwann cells or perineurial or endoneurial fibroblasts, respectively.

NI-1: a novel canine mastocytoma model for studying drug resistance and IgER-dependent mast cell activation.

BACKGROUND: Advanced mast cell (MC) disorders are characterized by uncontrolled growth of neoplastic MC in various organs, mediator-related symptoms, and a poor prognosis. Kit mutations supposedly contribute to abnormal growth and drug resistance in these patients. METHODS: We established a novel canine mastocytoma cell line, NI-1, from a patient suffering from MC leukemia. RESULTS: NI-1 cells were found to form mastocytoma lesions in NOD/SCID IL-2Rgamma(null) mice and to harbor several homozygous Kit mutations, including missense mutations at nucleotides 107(C→T) and 1187(A→G), a 12-bp duplication (nucleotide 1263), and a 12-bp deletion (nucleotide 1550). NI-1 cells expressed several MC differentiation antigens, including tryptase, Kit, and a functional IgE receptor. Compared to the C2 mastocytoma cell line harboring a Kit exon 11 mutation, NI-1 cells were found to be less responsive against the Kit tyrosine kinase inhibitors (TKI) masitinib and imatinib, but were even more sensitive against proliferation-inhibitory effects of the mammalian target of rapamycin (mTOR) blocker RAD001 and PI3-kinase/mTOR blocker NVP-BEZ235. The Kit-targeting multikinase inhibitors PKC412 and dasatinib were also found to override TKI resistance in NI-1 cells, and produced growth inhibition with reasonable IC(50) values (<0.1 µM). CONCLUSION: NI-1 may serve as a useful tool to investigate IgE-dependent reactions and mechanisms of abnormal growth and drug resistance in neoplastic MC in advanced mastocytosis.

Expression of toll-like receptor 2 mRNA in bronchial epithelial cells is not induced in RAO-affected horses.

REASONS FOR PERFORMING STUDY: Airway inflammation in recurrent airway obstruction (RAO) is triggered by housing affected horses in stables.It has been suggested that RAO is an allergic condition, but innate immune mechanisms are also involved. Fungal products activate innate immune mechanisms through toll-like receptor 2 (TLR2). In human airway epithelium, TLR2 activation leads to interleukin (IL)-8 production. This pathway is negatively regulated by the zinc finger protein A20. This study was performed to enhance understanding of innate immune mechanisms in RAO. HYPOTHESIS: TLR2 and IL-8 mRNA are elevated in RAO during stabling compared with controls. A20 mRNA is negatively associated with the numbers of airway inflammatory cells. OBJECTIVES: To determine TLR2, IL-8 and A20 mRNA expression in lungs of stabled and pastured RAO-affected and control horses. METHODS: Airway obstruction and inflammatory cell counts in bronchoalveolar lavage were measured, and TLR2, IL-8 and A20 mRNA expression quantified by qRT-PCR in 6 RAO-affected and 6 control horses, during and after exposure to hay and straw. RESULTS: Airway obstruction and neutrophils were increased in RAO-affected horses during stabling. While stabling increased IL-8, TLR2 and A20 mRNA were unaffected. TLR2 and A20 were significantly correlated (r = 0.83) and A20 mRNA was negatively associated with inflammatory cells. POTENTIAL RELEVANCE: Stabling does not lead to an increase in TLR2 expression. Other molecules or processes in the TLR2 cascade might be important in fungal-induced airway inflammation. Equine epithelial-derived A20 may be involved in modulation of airway inflammation.

Serum Cytokine Profiling in Cats with Acute Idiopathic Cystitis.

BACKGROUND: Feline idiopathic cystitis (FIC) is a common lower urinary tract disorder of domestic cats that resembles interstitial cystitis/painful bladder syndrome (IC/PBS) in humans. Diagnosis of FIC is based on clinical signs and exclusion of other disorders because of a lack of specific pathologic findings or other objective biomarkers. Cytokines are potential noninvasive biomarkers to define the presence, severity, and progression of disease, and response to treatment. OBJECTIVES: The objective of this pilot study was to determine concentrations of selected cytokines in serum from healthy cats and cats with acute FIC. ANIMALS: Serum samples from 13 healthy cats and from 12 cats with nonobstructive acute FIC were utilized. METHODS: Multiplex analysis of 19 cytokines (CCL2, CCL5, CXCL1, CXCL12, CXCL8, Flt3L, GM-CSF, IFN-γ, IL-12 (p40), IL-13, IL-18, IL-1ß, IL-2, IL-4, IL-6, PDGF-BB, SCF, sFas, and TNF-α) was performed with a commercially available feline-specific multiplex bead-based assay. RESULTS: Mean serum concentrations of IL-12 (p40; P < 0.0001), CXCL12 (P = 0.002), IL-18 (P = 0.032), and Flt3L (P = 0.0024) were significantly increased in FIC cats compared to healthy cats. GM-CSF, IL-1b, IL-2, and PDGF-BB were undetectable or detected in an insufficient number of cats to allow meaningful comparisons. CONCLUSIONS AND CLINICAL IMPORTANCE: We have identified increased serum concentrations of pro-inflammatory cytokines and chemokines CXCL12, IL-12, IL-18, and Flt3L in FIC-affected cats. These findings suggest potential candidates for noninvasive biomarkers for diagnosis, staging, and therapeutic outcome monitoring of affected cats and provide additional insight into the etiopathogenesis of FIC.

Gain-of-function mutation in PTPN11 in histiocytic sarcomas of Bernese Mountain Dogs.

Histiocytic sarcoma (HS) is an aggressive malignant neoplasm of dendritic cell origin that is common in certain breeds of dogs. High prevalence of fatal, disseminated HS has been described in Bernese Mountain Dogs (BMDs). Support for genetic predisposition to develop HS has been presented in several studies, but to date, causative genetic events have not been reported. In addition, no driver mutations have been identified in tumours. Recently, E76K gain-of-function mutation in SHP2 encoded by the PTPN11 gene has been described in human histiocytic malignancies. In our study, we identified the PTPN11E76K in HS of BMDs. Amplification of exon 3 of the PTPN11 gene followed by Sanger sequencing was used to detect the mutation and estimate the prevalence in HS from 30 BMDs, 13 Golden Retrievers and 10 other dog breeds. The overall prevalence of PTPN11E76K in HS of BMDs was 36.67% compared with 8.69% in other breeds. No mutation was identified in normal tissues from 10 BMDs with HS that carried the mutation and 12 control dogs with no neoplastic disease, including 6 BMDs. Increased immunoreactivity for AKT, phosphorylated ERK1/2 and phosphorylated AKT in a small subset of BMDs with PTPN11E76K suggests that a gain-of-function might be mediated by the ERK and AKT pathways. These data suggest PTPN11E76K as an important driver mutation of HS in BMDs. This information may not only aid in unravelling the tumourigenic events associated with HS in BMDs, but also help in identifying more promising therapeutic strategies.

Safety of Intraperitoneal Injection of Adipose Tissue-Derived Autologous Mesenchymal Stem Cells in Cats.

BACKGROUND: Chronic inflammatory diseases are common in cats and mesenchymal stem cells (MSC) are a promising therapeutic approach for management of these disorders. The purpose of this study was to evaluate the safety of intraperitoneal injection of MSC in cats. HYPOTHESIS: Intrapertioneal injection of autologous MSC in cats is safe. ANIMALS: Ten healthy adult purpose-bred cats. METHODS: Mesenchymal stem cells were isolated from subcutaneous adipose tissue collected during ovariohysterectomy and characterized for expression of CD90, CD105 and CD44 and trilineage differentiation. Three weeks postoperatively a complete blood count, serum chemistry profile, urinalysis, and abdominal ultrasound were performed. Five cats then received 1 × 10(6) of autologous MSC/kg of body weight intraperitoneally with ultrasound guidance; 5 additional cats were sham injected. Cats were monitored for 6 weeks with daily physical examinations and weekly clinicopathological evaluations. Abdominal ultrasonography was repeated at weeks 1 and 5 after injection. RESULTS: Serious adverse effects were not observed in any MSC-injected cat. Two animals developed transient lethargy and decreased activity. Jejunal lymph node size was increased in MSC-injected cats compared to controls at weeks 1 (1.38 ± 0.25 versus 0.88 ± 0.25 cm(2); P = .036) and 5 (1.75 ± 0.82 versus 0.79 ± 0.12 cm(2); P = .047). A hyperechoic renal segmental cortical lesion was observed in 1 MSC-injected cat. CONCLUSIONS AND CLINICAL RELEVANCE: Intraperitoneal MSC injection was well tolerated with only mild, self-limiting adverse effects being observed in 2 cats. This route provides a safe means of administration for cell-based treatment in cats.

Wilson disease.

Wilson disease is an inherited disorder of copper metabolism. Progress has been made in establishing the location of the gene on the long arm of chromosome 13, and in finding nearby probes that can be used to identify affected sibs of newly diagnosed patients. However, the gene has not been cloned, and the molecular nature of the defect remains unknown. The cause of the disease is a failure to excrete unneeded and excessive copper in the bile for loss in the stool. This may be due to a failure to excrete copper packaged in ceruloplasmin into the bile. Clinically, patients usually present during the second to fourth decades of life with liver, neurologic, or psychiatric disease, but the diagnosis is often missed or delayed. Once a diagnosis of Wilson disease is considered, reliable studies of copper variables can be carried out. After diagnosis, patients must receive anticopper treatment for the rest of their lives, to reduce copper levels and prevent copper reaccumulation. For life-long maintenance therapy, we recommend zinc acetate because of its complete efficacy and lack of toxicity; it acts by blocking copper absorption. For initial therapy of the acutely ill patient, no currently available therapy has proven to be ideal. A chelator-type drug, either penicillamine or trien, can be used for the initial therapy of patients who present with liver disease; transition to zinc acetate can then be made after a few months. For the initial therapy of acutely ill patients who present with neurologic disease, chelation should be avoided because neurologic worsening frequently occurs, probably due to redistribution of copper which temporarily raises the levels of copper in the brain. For initial treatment, zinc therapy is also not ideal because it is relatively slow-acting. A new experimental drug, tetrathiomolybdate, shows promise in the initial treatment of patients with Wilson disease. The major challenges ahead include closing the remaining therapeutic hiatuses, cloning and expressing the gene to understand its function, and improving clinical diagnosis so that therapy can be instituted as quickly as possible.

Treatment of Wilson's disease with ammonium tetrathiomolybdate. I. Initial therapy in 17 neurologically affected patients.

OBJECTIVE: To test the efficacy and toxicity of a new drug, ammonium tetrathiomolybdate, in the initial treatment of a relatively large series of patients presenting with neurologic signs and symptoms caused by Wilson's disease. The key aspect of efficacy was to preserve the neurologic function present at the onset of therapy. DESIGN: An open study of 17 patients treated for 8 weeks each. Neurologic function was evaluated by frequent quantitative neurologic and speech examinations. Several copper-related variables were studied to evaluate the effect of the drug on copper, and a large number of biochemical and clinical variables were studied to evaluate potential toxicity. Patients were then followed up at yearly intervals, with follow-up periods of 1 to 5 years reported. SETTING: A university hospital referral setting INTERVENTION: Patients were generally treated for 8 weeks with tetrathiomolybdate, followed by zinc maintenance therapy. MAIN OUTCOME MEASURES: Neurologic function was evaluated by quantitative neurologic and speech examinations. RESULTS: None of the patients suffered a loss of neurologic function. Copper status and potential further toxic effects were generally well controlled quickly. No toxic effects resulted from administration of tetrathiomolybdate. During the ensuing period of follow-up of 1 to 5 years, neurologic recovery in most patients was good to excellent. CONCLUSIONS: Tetrathiomolybdate appears to be an excellent form of initial treatment in patients with Wilson's disease presenting with neurologic signs and symptoms. In contrast to penicillamine therapy, initial treatment with tetrathiomolybdate does not result in further, often irreversible neurologic deterioration.

Characterization of eae+ Escherichia coli isolated from healthy and diarrheic calves.

Strains of Escherichia coli from 101 healthy and 114 diarrheic calves were screened by PCR for the eae (intimin) gene and Shiga toxin genes (stx). Each eae+ and eae/stx+ strain was examined for antimicrobial susceptibility, enterohemolysin activity, and the somatic O antigen was determined. An immunoassay was used to detect Shiga toxin antigens for the eae/stx+ E. coli. Significantly more (p = 0.005) of the healthy calves carried eae+ and eae/stx+ E. coli in their feces when compared to strains from diarrheic calves. Moreover, Shiga toxin antigens were detected significantly more (p = 0.001) often among the eae/stx+ strains from healthy calves when compared to eae/stx+ strains from diarrheic calves. However, significantly more (p = 0.001) of the eae+ and eae/stx+ strains from diarrheic calves were resistant to at least one of the antimicrobials tested, and the strains from diarrheic calves had a significantly (p = 0.05) higher rate of antimicrobial resistance to at least two different antimicrobial classes. No significant difference (p> or =0.05) was detected among the eae+ and eae/stx+ strains from healthy and diarrheic calves for enterohemolysin production. Serogroups O-negative, O5, O26, and O111 were predominate among both healthy and diarrheic calves.

Treatment of Wilson's disease with zinc XII: dose regimen requirements.

A considerable body of data is now available indicating the efficacy and lack of toxicity of zinc treatment of Wilson's disease. Dose-response studies have shown that regimens of 50 mg of elemental zinc 3 times a day (50 mg x 3), 25 mg x 3, and 50 mg x 2 are effective, but 25 mg x 2 and 50 mg x 1 are not adequately effective. These studies indicate that 75 mg a day is close to the minimally effective dose, but do not address the question of necessary dose frequency. In the current study, the authors have used the minimally effective daily dose, 75 mg, and studied this daily dose in regimens of 25 mg x 3, 37.5 mg x 2, and 75 mg x 1 in treatment of four patients with Wilson's disease. These data have been supplemented with additional data from 11 patients treated with 25 mg 3 times a day and with data from 2 patients treated with 75 mg once a day. Efficacy was evaluated by 10-day copper balance and absorption of orally administered 64copper. The findings indicate that a daily dose of 75 mg must be divided into at least two doses to be effective, and that the 64copper procedure is more sensitive to zinc dose than copper balance.

Characterization of an undifferentiated malignancy as a mast cell tumor using mutation analysis in the proto-oncogene c-KIT.

A 6.5-year-old female Boxer was euthanized and presented for necropsy following rapid clinical decline concomitant with the development of numerous tumor masses. The largest of these masses was in the same location as a mast cell tumor that had been previously removed from this dog. Gross examination revealed the presence of nodules 5-200 mm in diameter throughout the body, including the lymph nodes. Histologic analysis showed an influx of round cells with no granules, leading to the provisional diagnosis of systemic lymphosarcoma. Immunohistochemical staining for B- and T-lymphocyte antigens was negative. Molecular tests were used to identify a tandem duplication in the c-KIT proto-oncogene from both the earlier mast cell tumor and the current nodules, implicating a common origin. Addition of molecular testing to conventional necropsy evaluations allowed a definitive diagnosis of mast cell tumors.

Bovine hereditary zinc deficiency: lethal trait A 46.

Bovine hereditary zinc deficiency, also referred to as Adema disease, is an autosomal recessive disorder which results in inadequate amounts of zinc being absorbed from the gastrointestinal tract and leads to a number of clinical abnormalities. Using semen from a homozygous affected bull and obligate heterozygote cows in embryo transfer studies, 7 offspring were obtained. These included 5 affected calves and 1 heterozygous carrier; the seventh calf died within 48 hours of birth undiagnosed. One unaffected, unrelated bull calf was raised as a control. All the calves were raised and maintained under similar management conditions designed to minimize secondary complications that would obscure the clinical and biochemical observations of a zinc deficient state. The first clinical manifestation of zinc deficiency was diarrhea, followed by skin lesions, poliosis, and a decreased ability to sustain a suckle reflex. Trace mineral analysis of plasma blood samples revealed that plasma zinc concentrations of all the calves were normal at birth; however, they gradually declined in affected calves over the course of 3-8 weeks postpartum to below 0.5 ppm. Biochemical analysis of serum samples showed alkaline phosphatase activity consistently paralleled changes in the plasma zinc concentrations. The oral administration of zinc acetate caused a reversal of all clinical, biochemical, and histologic abnormalities in affected calves. The study of these affected calves allows further insight into the biological role of zinc as well as provides an animal model for the continued investigation of the human homologue acrodermatitis enteropathica.

Use of 64copper measurements to diagnose canine copper toxicosis.

Inherited canine copper toxicosis is a serious problem in Bedlington terriers and West Highland White terriers, and may also be a problem in other less-studied breeds. Affected dogs become ill at midlife with progressive and ultimately fatal liver disease. Treatments for removal of copper and prevention of copper accumulation are available, but are most effective if begun before the dog becomes ill. Until recently diagnosis has not been available until the dog is 1 year of age, and then only by an invasive liver biopsy with determination of liver copper concentration. The authors studied the use of 64copper for early diagnosis of canine copper toxicosis. Two procedures were evaluated. The first involved measuring the concentration of 64copper in blood 24 hours after oral administration of the radioisotope. At this time, 64copper was associated primarily with ceruloplasmin secreted into the blood by the liver. This procedure is useful in the diagnosis of the human counterpart, Wilson's disease. However, the authors found it to be nondiscriminatory between affected and unaffected dogs. In contrast, the second procedure, which involved measuring 64copper excreted in stool during 48 hours after an intravenous dose of radioisotope, yielded results that differentiated most affected and unaffected dogs.

Mutation causing von Willebrand's disease in Scottish Terriers.

Von Willebrand's Disease (vWD) in the Scottish Terrier breed is a serious, often fatal, hereditary bleeding disorder. Elimination of the mutated gene by selective breeding is an important goal for the health of this breed. Although the standard protein-based tests are accurate for identification of affected Scottish Terriers, they are not reliable for the identification of carriers of the mutant gene unless multiple replicate assays are performed. A simple, highly accurate test for carriers of the disease is needed so that veterinarians can counsel clients on which animals to use in their breeding programs. The complete coding region of von Willebrand factor (vWF) complementary DNA (cDNA) was sequenced from an affected animal, and a single base deletion in the codon for amino acid 85 of the prepro-vWF cDNA that leads to Scottish Terrier vWD was identified. A highly accurate polymerase chain reaction assay was developed that can distinguish homozygous normal animals from those that are homozygous affected or heterozygous. In a voluntary survey of 87 animals provided by Scottish Terrier owners, 15 were carriers and 4 were affected with vWD, 2 of which had previously been shown to have undetectable vWF. The determination of the complete canine vWF cDNA sequence should facilitate the identification of additional vWD alleles in other breeds and other species.

Population dynamics of inherited copper toxicosis in Dutch Bedlington terriers (1977-1997).

Inherited copper toxicosis in Bedlington terriers was 1st reported in 1975 and the entire Dutch population was examined from 1976 until the present for presence of the disease. To examine the effect on the prevalence of the disease of excluding affected dogs from breeding we have compared 2 time cohorts, the 1st consisting of dogs born from January 1, 1976, to January 1, 1986 (n = 155), and the 2nd of dogs born from January 1, 1990, to January 1, 1997 (n = 195). The diagnosis was made in the 1st cohort by evaluating liver biopsies, and in the 2nd cohort with a DNA marker. The population was also resolved into clusters of related dogs to analyze the familial distribution of the disease in the population and to search for ancient founders of the disease among the ancestors of sick dogs. Forty-six percent of dogs examined between 1976 and 1986 had copper toxicosis. Eleven percent of dogs examined in the 2nd cohort had evidence of disease. This reduction was achieved while maintaining the already limited genetic heterogeneity of the population: the number of clusters and the mean relatedness between the clusters were similar in both time cohorts. The disease was evenly distributed over the clusters of related dogs in both cohorts. All ancestors had contributed to the distribution of copper toxicosis and no specific founders could be identified. This indicates that when the breed was established in The Netherlands, the disease was already highly prevalent in the founding dogs.

Recombinant equine interleukin-1beta induces putative mediators of articular cartilage degradation in equine chondrocytes.

Interleukin-1 is considered a central mediator of cartilage loss in osteoarthritis in several species, however an equine recombinant form of this cytokine is not readily available for in vitro use in equine osteoarthritis research. Equine recombinant interleukin-1beta was cloned and expressed and its effects on the expression and activity of selected chondrocytic proteins implicated in cartilage matrix degradation were characterized. Reverse transcriptase polymerase chain reaction methods were used to amplify the entire coding region of the equine IL-1beta mRNA, which was cloned into an expression vector, expressed in E. coli, and purified using a Ni2+ chromatographic method. The effects of the recombinant peptide on chondrocyte gene expression were determined by Northern blotting using RNA from equine chondrocyte cultures hybridized to probes for matrix metalloproteinases (MMP 1, MMP 3, MMP 13), tissue inhibitor of matrix metalloproteinases 1 (TIMP 1) and cyclooxygenase 2 (COX 2). Effects on selected mediators of cartilage degradation (nitrite concentrations and MMP activity) were determined using conditioned medium from reIL-1beta-treated equine cartilage explant cultures. A recombinant peptide of approximately 21 kd was obtained. Northern blotting analyses revealed a marked up-regulation of expression of all MMPs, TIMP 1, and COX 2 in mRNA from treated chondrocytes. Furthermore, cartilage explants exposed to reIL-1beta had augmented collagenase/gelatinase and stromelysin activities as well as increased concentration of nitrite in conditioned media. The development of a biologically active, species-specific IL-1beta provides a valuable tool in the study of osteoarthritis pathophysiology and its treatment in horses.

Development and usefulness of new polymerase chain reaction-based tests for detection of different alleles at codons 136 and 171 of the ovine prion protein gene.

OBJECTIVE: To develop new and improved tests to detect alleles at codons 136 and 171 of the ovine prion protein locus and to evaluate the frequency of these alleles. ANIMALS: 159 Suffolk sheep belonging to 3 flocks. PROCEDURE: Polymerase chain reaction (PCR) analysis that contained diagnostic restriction site variation for each allele were developed for the relevant gene regions. Alleles were determined by analyzing DNA isolated from buccal swab specimens or blood samples. RESULTS: At codon 136, frequencies of the alanine and valine alleles were found to be 97 and 3%, respectively. At codon 171, frequencies of the glutamine, arginine, and histidine alleles were found to be 57, 41, and 2%, respectively. CONCLUSIONS: Little variation was detected in codon 136, whereas noteworthy variation was found in codon 171; > 40% of the alleles at this locus coded for glutamine. Because the glutamine allele at codon 171 confers susceptibility to scrapie, reduction of its frequency is of importance to management of sheep flocks. CLINICAL RELEVANCE: Genotyping of sheep, using the tests reported here, should facilitate selective breeding programs designed to decrease the risk of scrapie.

Linkage of a microsatellite marker to the canine copper toxicosis locus in Bedlington terriers.

OBJECTIVE: To identify a DNA marker for the copper toxicosis (CT) locus in Bedlington Terriers (BT). ANIMALS: 77 BT, of which 25 were affected. Diagnosis of affected or unaffected with CT was made in all cases by quantitative copper determinations on liver biopsy samples by use of established criteria. PROCEDURE: BT pedigrees segregating for CT were identified. Linkage studies were carried out using polymorphic microsatellite markers developed for the canine genome in these pedigrees. DNA was isolated from blood samples of pedigree members. Polymerase chain reaction was used to amplify and type alleles at 213 microsatellite loci in each dog, and findings were subjected to linkage analysis. RESULTS: One microsatellite marker was identified to be closely linked to CT with logarithm of odds score of 5.96 at a recombination fraction of zero. CONCLUSIONS: Using the linked marker, it has become possible to distinguish affected, homozygous normal, and carrier dogs in some BT pedigrees. CLINICAL RELEVANCE: In informative pedigrees where the marker is variable in the parents, it is possible to identify which dogs will require anticopper therapy and provide breeders with sound scientific advice about breeding strategies.

Use of zinc acetate to treat copper toxicosis in dogs.

Zinc acetate was used for the treatment and prophylaxis of hepatic copper toxicosis in 3 Bedlington Terriers and 3 West Highland White Terriers. Two dogs of each breed were treated for 2 years, and 1 of each breed for 1 year. A dosage of 200 mg of elemental zinc per day was required to achieve therapeutic objectives related to copper, which included a doubling of plasma zinc concentration to 200 micrograms/dl and a suppression of oral 64 copper absorption. The dosage was later reduced to 50 to 100 mg/day to avoid an excessive increase in plasma zinc concentration. The preliminary clinical results were good. Three dogs had mild to moderate active liver disease and high liver copper concentrations at the time of initiation of zinc administration. Biopsy of the liver 2 years later revealed a reduction in hepatitis and copper concentrations. One other dog without active hepatitis also had a reduction in hepatic copper concentrations over a 2-year period. All 6 dogs have done well clinically. On the basis of these findings, we believe zinc acetate to be an effective and nontoxic treatment for copper toxicosis in dogs.