Survival time for dogs with previously untreated, peripheral nodal, intermediate- or large-cell lymphoma treated with prednisone alone: the Canine Lymphoma Steroid Only trial.

OBJECTIVE: To evaluate survival times for dogs with previously untreated, peripheral nodal, intermediate- or large-cell lymphoma treated with prednisone alone. ANIMALS: 109 client-owned dogs recruited from 15 institutions in the United States. PROCEDURES: Dogs were treated with prednisone at a dosage of 40 mg/m2, PO, once daily for 7 days and at a dosage of 20 mg/m2, PO, once daily thereafter. Quality of life (QOL) was assessed by owners with a visual analog scale when treatment was started (day 0), 1 and 2 weeks after treatment was started, and every 4 weeks thereafter. The primary outcome of interest was survival time as determined by the Kaplan-Meier method. Factors potentially associated with survival time were examined. RESULTS: Median overall survival time was 50 days (95% CI, 41 to 59 days). Factors associated with survival time included substage (a vs b) and immunophenotype (B cell vs T cell). Owner-assigned QOL scores on days 0 and 14 were significantly positively correlated with survival time. When QOL score was dichotomized, dogs with day 0 or day 14 QOL scores ≥ 50 had significantly longer survival times, compared with dogs with day 0 or day 14 QOL scores < 50. No variables were predictive of long-term (> 120 days) survival. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggested that survival times were short for dogs with previously untreated, peripheral nodal, intermediate- or large-cell lymphoma treated with prednisone alone. Owner-perceived QOL and clinician-assigned substage were both associated with survival time. Findings provide potentially important information for clinicians to discuss with owners of dogs with lymphoma at the time treatment decisions are made.

Identification of blue staining vaccine-derived material in inflammatory lesions using cultured canine macrophages.

BACKGROUND: Vaccine reactions are described in cytology textbooks as having eosinophilic to magenta colored globules within and admixed with inflammatory cells. Recently, we have seen increased numbers of inflammatory lesions containing blue to blue-gray globular material, with historical information suggesting an association with rabies vaccination. OBJECTIVES: The purpose of the study was to confirm the blue-gray and the eosinophilic material observed microscopically in some inflammatory lesions as being vaccine-derived. METHODS: Three different vaccines were cytocentrifuged and Wright stained. Vaccine aliquots were also added to the culture media of canine-derived macrophages for 24 hours and the cells subsequently harvested, cytocentrifuged, and Wright stained. The globular material present in both preparations was compared to that observed in vaccine-induced inflammatory lesions. Morin staining was used to identify metal within vaccine material in both in vitro- and in vivo-derived cytology samples. RESULTS: Vaccine-derived material has a characteristic color and appearance. Appearance of the material was consistent in cytologic samples, in cells incubated with the vaccine, and in cytocentrifuged preparations of the vaccine vial contents. The blue-gray globules stained positively for Morin stain, while the eosinophilic material did not stain. CONCLUSIONS: Vaccine-induced inflammatory lesions may contain blue to blue-gray or magenta stained globular material. Blue-gray material was associated with administration of rabies vaccine Imrab 3 TF and the observed material may be metal-containing adjuvant. Magenta material was associated with other vaccines and negative for Morin stain, suggesting a metal-free adjuvant.

May-Hegglin anomaly in a dog.

An 8-year-old female spayed Pug dog was presented for evaluation of cutaneous lesions occurring secondary to immunosuppressive treatment of presumed immune-mediated thrombocytopenia. Abnormal hematologic findings included persistent thrombocytopenia, macrothrombocytes, and variably shaped, often fusiform, blue cytoplasmic inclusions in neutrophils. May-Hegglin anomaly (MHA) was suspected based on the morphologic appearance of platelets and neutrophils. Examination of cells by transmission electron microscopy revealed normal platelet ultrastructure; neutrophil inclusions had features similar to those reported for inclusions in human MHA. Neutrophil function was within normal limits based on flow cytometric analysis. Thrombelastography indicated a prolonged clotting time (r), and PlateletMapping showed a lack of response to 2 µM ADP compared with a moderate response in the control dog. Immunocytochemical staining of blood smears using 2 commercially available antibodies against MYH9 protein (nonmuscle myosin heavy chain II) yielded negative results. However, genomic DNA sequencing analysis of the dog's MYH9 gene identified a single point mutation, resulting in substitution of lysine for glutamine at the 1841 amino acid position; this mutation is identical to one identified in people with MHA. To our knowledge, this is the first report of an MYH9 mutation in the dog. MHA-associated macrothrombocytopenia may be mistaken for immune-mediated thrombocytopenia.

Evaluation of peripheral blood neutrophil function in tumor-bearing dogs.

BACKGROUND: Peripheral blood neutrophils of untreated human cancer patients have been shown to have normal, increased, and decreased phagocytic activity, killing capacity, and/or oxidative burst activities. OBJECTIVES: The objectives of this study were to evaluate oxidative burst and phagocytic activities of peripheral blood neutrophils from tumor-bearing dogs before therapy and compare them with neutrophil function of healthy control dogs. METHODS: Heparinized whole blood was obtained from dogs with high-grade lymphoma (n=23), sarcoma (n=13), or carcinoma (n=11), and healthy control dogs (n=11) for flow cytometric evaluation of oxidative burst and phagocytic activities. Percentage of bursting cells and amount of oxidative burst activity were determined after stimulation with phorbol 12-myristate 13-acetate (PMA) or Escherichia coli. Percentage of phagocytic cells and amount of phagocytic activity were determined after incubation with fluorescent E. coli. RESULTS: Compared with control dogs, dogs with sarcoma (P=.004) and carcinoma (P=.05) had a lower percentage of neutrophils exhibiting oxidative burst activity after stimulation with PMA. Phagocytic activity was significantly lower in dogs with sarcomas compared with control dogs (P<.0001) and dogs with lymphoma (P=.01). CONCLUSIONS: Untreated carcinomas and sarcomas in dogs may suppress the percentage of neutrophils capable of oxidative burst when stimulated by PMA. Furthermore, sarcomas also may suppress the amount of phagocytic activity per neutrophil. Until further studies can be performed, the clinical significance of these findings is unknown.

Comparison of aspiration and nonaspiration techniques for obtaining cytologic samples from the canine and feline spleen.

BACKGROUND: Ultrasound-guided fine needle aspiration of the spleen is commonly used in the diagnostic evaluation of veterinary patients. Techniques using suction delivered through a 6-20-cm(3) syringe are the most commonly described means of obtaining cytologic samples of the spleen. Comparison studies of various human lesions have shown nonaspiration techniques to produce equal or superior cytologic specimens with less blood than specimens obtained using aspiration techniques. OBJECTIVE: The purpose of this study was to compare the quality of splenic cytology specimens obtained using aspiration and nonaspiration techniques. METHODS: Client-owned dogs (n=24) and cats (n=7) receiving an abdominal ultrasound at the University of Tennessee College of Veterinary Medicine were enrolled in the study between January and June 2005. Samples were obtained from patients with and without sonographic splenic abnormalities. Two clinical pathologists, working independently and blinded to the method of sample collection, graded the cytologic specimens using a subjective scoring system for cellularity, amount of blood, and preservation of cellular morphology. RESULTS: Agreement between the 2 independent observers was good. Direct comparison of the 2 techniques showed that samples obtained by the nonaspiration method had higher cellularity (P=.0002), less blood (P=.0023), and similar cell morphology (P=1.0000) compared with samples obtained by the aspiration method. CONCLUSION: These results suggest the nonaspiration technique is a superior method for obtaining a high-quality cytologic specimen from the canine and feline spleen.

Flow cytometric evaluation of multidrug resistance proteins on grossly normal canine nodal lymphocyte membranes.

OBJECTIVE: To demonstrate efficacy of flow cytometric evaluation of expression and activity of P-glycoprotein (P-gp) and multidrug resistance-associated protein (MRP) efflux pumps and characterize and correlate their expression and activity in grossly normal canine nodal lymphocytes. SAMPLE POPULATION: Nodal lymphocytes from 21 clinically normal dogs. PROCEDURES: Pump expression was assessed by use of fluorescent-labeled mouse antihuman P-gp (C494) and MRP1 (MRPm6) antibodies and expressed as median values (antibody value divided by isotype control value). The P-gp and MRP activities were assessed by measuring cellular retention of rhodamine 123 and 5(6)-carboxyfluorescein diacetate in the absence and presence of inhibitors (verapamil and PSC833 for P-gp, probenecid and MK-571 for MRP). Protein activity was expressed as median fluorescence of cells with inhibitors divided by that without inhibitors. RESULTS: Expression of P-gp was (mean +/- SEM) 50.62 +/- 13.39 (n = 21) and that of MRP was 2.16 +/- 0.25 (13). Functional activity was 1.27 +/- 0.06 (n = 21) for P-gp and both inhibitors and 21.85 +/- 4.09 (21) for MRP and both inhibitors. Function and expression were not correlated. CONCLUSIONS AND CLINICAL RELEVANCE: Use of flow cytometry effectively assessed P-gp and MRP expression and activity in canine lymphocytes. Optimization of the flow cytometric assay was determined for evaluating activity and expression of these pumps in canine lymphoid cells. Evaluation of expression or activity may offer more meaning when correlated with clinical outcome of dogs with lymphoproliferative diseases. Cell overexpression of P-gp and MRP can convey drug resistance.

Effects of dietary supplementation with fish oil on in vivo production of inflammatory mediators in clinically normal dogs.

OBJECTIVE: To evaluate the effect of diets enriched with eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) on in vivo production of interleukin (IL)-1, IL-6, tumor necrosis factor (TNF)-alpha, prostaglandin E2 (PGE2), and platelet-activating factor (PAF) in dogs. ANIMALS: 15 young healthy dogs. PROCEDURES: Dogs were randomly allocated to receive an isocaloric ration supplemented with sunflower oil (n=5), fish oil (5), or fish oil plus vitamin E (5) for 12 weeks. At week 12, in vivo production of inflammatory mediators was evaluated in serum at multiple time points for 6 hours following stimulation with IV administration of lipopolysaccharide (LPS). RESULTS: Serum activity or concentration (area under the curve) of IL-1, IL-6, and PGE2 significantly increased after LPS injection in all groups but to a lesser extent in dogs receiving the fish oil diet, compared with results for dogs receiving the sunflower oil diet. Serum activity of TNF-alpha and PAF concentration also increased significantly after LPS injection in all groups but did not differ significantly among groups. CONCLUSIONS AND CLINICAL RELEVANCE: A fish oil-enriched diet consisting of 1.75 g of EPA/kg of diet and 2.2 g of DHA/kg of diet (dry-matter basis) with an n-6:n-3 fatty acid ratio of 3.4:1 was associated with significant reductions in serum PGE2 concentrations and IL-1 and IL-6 activities. Results supported the use of EPA- and DHA-enriched diets as part of antiinflammatory treatments for dogs with chronic inflammatory diseases. Additional studies in affected dogs are warranted to further evaluate beneficial anti-inflammatory effects of EPA- and DHA-enriched diets.

Hypercalcemia associated with gastric pythiosis in a dog.

A 20-month-old castrated male Labrador Retriever with a 3-month history of anorexia, weight loss, and vomiting was evaluated. Plasma biochemical abnormalities included marked hyperglobulinemia and hypercalcemia. Serum levels of parathyroid hormone, parathyroid hormone-related protein, 25-hydroxyvitamin D, and 1,25-dihydroxyvitamin D were either low or within reference intervals. Gastric wall thickening and abdominal lymphadenomegaly were observed with abdominal ultrasonography. Cytologic evaluation of a sample obtained via fine-needle aspiration of the gastric wall revealed pyogranulomatous inflammation and numerous poorly stained hyphae. Partial gastrectomy was performed, and a diagnosis of gastric pythiosis was made by immunohistochemical staining of infected gastric tissue, as well as by immunoblot serology. This case demonstrates that diagnostic samples for cytologic evaluation can be obtained by fine-needle aspiration of Pythium insidiosum-infected tissues and that a presumptive diagnosis can be made by examination of a Romanowsky-stained smear. Furthermore, pythiosis should be considered as a differential diagnosis for hypercalcemia, especially in young dogs with inflammatory lesions that have a granulomatous component. The mechanism for the hypercalcemia in this dog was not determined; however, calcium concentrations normalized after surgical resection of the gastric lesion.

Cerebral Blastomyces dermatitidis infection in a cat.

CASE DESCRIPTION: An 8-year-old domestic shorthair cat was evaluated because of signs of depression, circling, and visual deficits. CLINICAL FINDINGS: The cat had no cutaneous lesions, and results of an ophthalmologic examination and thoracic radiography were within reference limits. Computed tomography of the brain revealed a mass lesion involving the right parietal, temporal, and occipital lobes; the mass was in broad-based contact with the skull and smoothly marginated and had strong homogenous enhancement after contrast agent administration. During craniectomy, samples of the mass were collected for cytologic and histopathologic evaluations and microbial culture. A diagnosis of Blastomyces dermatitidis-associated meningoencephalitis with secondary pyogranulomatous inflammation was made. TREATMENT AND OUTCOME: Amphotericin B (0.25 mg/kg [0.11 mg/lb], IV) was administered on alternate days (cumulative dose, 1.75 mg/kg [0.8 mg/lb]). To minimize the risk of nephrotoxicosis, assessments of serum biochemical variables (urea nitrogen and creatinine concentrations) and urinalyses were performed at intervals. The third dose of amphotericin B was postponed 48 hours because the cat became azotemic. The cat subsequently received fluconazole (10 mg/kg [4.5 mg/lb], PO, q 12 h) for 5.5 months. Six months after discontinuation of that treatment, the cat appeared healthy and had no signs of relapse. CLINICAL RELEVANCE: Brain infection with B dermatitidis is typically associated with widespread disseminated disease. The cat of this report had no evidence of systemic disease. Blastomycosis of the CNS should be considered as a differential diagnosis for brain lesions in cats from areas in which B dermatitidis is endemic.

Effects of dietary fish oil and vitamin E supplementation on canine lymphocyte proliferation evaluated using a flow cytometric technique.

Lymphocyte proliferation and peripheral blood mononuclear cell (PBMC) production of PGE(2) were assayed in 15 healthy dogs fed a basal diet supplemented with either sunflower oil (Group Sunflower oil), sunflower oil and menhaden fish oil (Group Fish oil), or sunflower oil and menhaden fish oil plus alpha-tocopherol acetate for 12 weeks (Group Fish oil + E). Lymphocyte proliferation was determined by a flow cytometric technique utilizing the fluorochrome carboxyfluorescein diacetate succinimidyl ester (CFSE). The PBMC supernatant PGE(2) concentration was assayed using a competitive enzyme-linked immunoassay. Group Fish oil had a significant decrease in lymphocyte proliferation at week 12. PBMC production of PGE(2) was decreased in all three groups but only significantly reduced in groups receiving fish oil supplementation. Based on these results, this level of fish oil supplementation appears to suppress the lymphoproliferative response in healthy, young dogs but this response can be attenuated by high levels of dietary vitamin E supplementation. Furthermore, fish oil-induced reduction in lymphocyte proliferation appears to manifest through a PGE(2)-independent mechanism and is not associated with increased lipid peroxidation.

Serial alterations in digital hemodynamics and endothelin-1 immunoreactivity, platelet-neutrophil aggregation, and concentrations of nitric oxide, insulin, and glucose in blood obtained from horses following carbohydrate overload.

OBJECTIVE: To quantify changes in endothelium-derived factors and relate those changes to various aspects of digital hemodynamics during the prodromal stages of carbohydrate overload (CHO)-induced laminitis in horses. ANIMALS: 20 adult horses without abnormalities of the digit. PROCEDURES: Digital and jugular venous blood samples were collected at 1-hour intervals (for assessment of endothelin-1 [ET-1] immunoreactivity and measurement of glucose, insulin, and nitric oxide [NO] concentrations) or 4-hour intervals (CBC and platelet-neutrophil aggregate assessment) for 8 hours or 16 hours after induction of CHO-associated laminitis in horses treated with an ET-1 antagonist. Effects of treatment, collection site, and time and the random effects of horse on each variable were analyzed by use of a repeated-measures model. Where treatment and collection site had no significant effect, data were combined. RESULTS: Compared with baseline values, CHO resulted in changes in several variables, including a significant increase from baseline in digital blood ET-like immunoreactivity at 11 hours; digital blood ET-like immunoreactivity was significantly greater than that in jugular venous blood at 8, 9, 11, and 12 hours. Digital and jugular venous blood concentrations of glucose increased from baseline significantly at 3, 4, and 5 hours; insulin concentration increased significantly at 5 hours; and the number of platelet-neutrophil aggregates increased significantly at 12 hours. CONCLUSIONS AND CLINICAL RELEVANCE: In horses, concurrent increases in venous blood ET-1 immunoreactivity, insulin and glucose concentrations, and platelet-neutrophil aggregates support a role of endothelial dysfunction in the pathogenesis of CHO-induced laminitis.

Effect of dietary fish oil and vitamin E supplementation on hematologic and serum biochemical analytes and oxidative status in young dogs.

Fifteen healthy dogs received a basal diet supplemented with either 12.4 g of sunflower oil, 0.6 g of sunflower oil and 7 g of menhaden fish oil, or 0.6 g of sunflower oil and 7 g of menhaden fish oil plus 0.18 g of alpha-tocopherol acetate for twelve weeks. There was no significant diet effect on platelet aggregation, lipid peroxidation, or standard hematologic and biochemical parameters, with the exception of decreased triglycerides in dogs supplemented with fish oil. These data demonstrate that this level of fish oil supplementation in dogs does not require vitamin E supplementation above recommended dosage and may prove beneficial in the treatment of hyperlipidemia in the dog.

Firm rib mass aspirate from a dog.

A 9-year-old intact male Miniature Schnauzer presented laterally recumbent, cachexic, and dehydrated with multiple firm bone masses and a bilaterally enlarged prostate. Fine-needle aspiration of a rib mass revealed numerous basophilic polygonal to fusiform cells predominantly found in small to large clusters. The cells exhibited cytologic criteria of malignancy and infrequently displayed large cytoplasmic vacuoles containing finely- to coarsely-stippled azurophilic material. The cytologic diagnosis was metastatic adenocarcinoma and was suspected to be prostatic or transitional cell in origin because of the azurophilic vacuoles within malignant cells. Gross and histologic findings confirmed the clinical and cytologic diagnosis of prostatic adenocarcinoma with widespread metastasis. Cytochemical and immunohistochemical investigation confirmed glycogen was a component of the vacuolar material. The vacuoles observed in the tumor in this case are not a consistent finding in tumors of the prostate or urinary bladder; however, when associated with an epithelial tumor, they may aid in limiting the differentials of the primary tumor and in the selection of further diagnostics.

Passive sampling screening method using thin-layer chromatography plates.

An in situ solid-phase extraction method, thin-layer chromatography (TLC) passive sampling, was investigated as a screening method for determining the presence of organic compounds in water using laboratory experiments and field applications. The TLC passive sampler developed in this research enables the spatial and temporal distributions of organic compounds to be determined qualitatively with little expense. The materials for the sampler developed here cost dollar 1.60 each. By first identifying the areas where a pollutant is present using the TLC passive sampling screening method, total analytical costs for monitoring programs may be reduced by eliminating unnecessary conventional analyses for locations where the pollutant is not present. Two organophosphorus pesticides, diazinon and chlorpyrifos, were used as a model for the development and as a potential application of the TLC passive sampling method. Passive sampler adsorption kinetics, enrichment factors, and extraction efficiencies were determined from batch experiments with exposure times ranging from 1 h to 4 weeks. In field applications, TLC passive sampling was conducted in natural and engineered systems with two silica gel extraction media, C2 and C18. Diazinon and chlorpyrifos were detected by analyzing the adsorbed compounds by high-performance liquid chromatography with ultraviolet detection.