Effects of Atorvastatin and Rosuvastatin on Blood Lipids in Quaker Parrots (Myiopsitta monachus).

Statin drugs are the most effective class of hypolipidemic and antiatherosclerotic drugs, with atorvastatin and rosuvastatin being the most effective. While the use of statins would be a tremendous asset in the treatment of dyslipidemia and lipid-accumulation disorders in birds, there are only limited data available regarding their use and effectiveness in psittacine species. Two consecutive randomized crossover trials on Quaker parrots (Myiopsitta monachus) were performed to study the effect of atorvastatin and rosuvastatin. Ten birds were used in an initial balanced crossover experiment with 5 oral treatments (control; atorvastatin 10 mg/kg q12h and q24h; rosuvastatin 10 mg/kg q12h and q24h) for 2 weeks each. Plasma lipidomics and lipoprotein profiling were performed after each treatment. Twelve birds were used in a second experiment consisting of 2 parallel crossover studies, each with 6 birds either fed their regular diet or a 0.3% cholesterol diet. In the 2 parallel crossover studies, the treatment group was administered atorvastatin 20 mg/kg orally q12h and the control group a placebo suspension orally q12h. Plasma lipidomics, lipoprotein profiles, and 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase activity were subsequently measured. Results were analyzed with serial linear mixed models and trends were assessed graphically. No statistically significant effect of any statin treatment was detected on plasma lipids, lipoproteins, creatinine kinase, or HMG-CoA reductase activity. In the first trial, all the rosuvastatin treatments led to some nonsignificant decreases in several triacylglycerol species, while in the second trial this was only observed in the birds on atorvastatin 20 mg/kg q12h being fed their regular diet. Quaker parrots may require much higher doses of statin drugs to show significant and clinically useful lipid-lowering effects.

Neonatal hyperleukocytosis and regenerative anemia in a septic puppy.

This paper reports a case of neonatal hyperleukocytosis in a dog due to a bacterial infection. A 3-week-old, mixed-breed dog was brought to a veterinary college referral center with a history of weight loss despite a good appetite. Clinical and laboratory examinations included: physical examination, complete blood (cell) count (CBC), serum biochemistry profile, abdominal ultrasound examination, and cytology of liver and bone marrow aspirates. The CBC showed hyperleukocytosis of 158.0 × 109/L (RI: 2.1 to 21.2 × 109/L) and hematocrit of 0.19 L/L (RI: 0.21 to 0.34 L/L). The strong leukemoid reaction was comprised of neutrophils, monocytes, and lymphocytes. The dog was diagnosed with Staphylococcus pseudointermedius liver infection based on liver aspirates and culture. Amoxicillin-clavulanic acid was prescribed. A recheck abdominal ultrasound and CBC repeated 4 wk after initial examination were unremarkable. Neonatal hyperleukocytosis is well-described in human medicine but veterinary studies in small animal neonates are scarce. Key clinical message: Hyperleukocytosis in adult dogs may be caused by leukemia or leukemoid reactions. Generalized sepsis is a leading cause of leukemoid reactions in adult dogs and cats. In puppies, neoplasia is less likely, and other causes should be investigated. Similar to human neonates, puppies can mount a strong leukemoid reaction during an infection, even if it is not a generalized septic process.

Hyperleucocytose néonatale et anémie régénérative chez un chiot septique. Cet article rapporte un cas d'hyperleucocytose néonatale chez un chien dû à une infection bactérienne. Un chien de race mixte âgé de 3 semaines a été amené dans un centre de référence d'une école vétérinaire avec des antécédents de perte de poids malgré un bon appétit. Les examens cliniques et de laboratoire comprenaient : examen physique, numération globulaire complète (CBC), profil biochimique sérique, examen échographique abdominal et cytologie des aspirations du foie et de la moelle osseuse.Le CBC montrait une hyperleucocytose de 158,0 × 109/L (RI : 2,1 à 21,2 × 109/L) et un hématocrite de 0,19 L/L (RI : 0,21 à 0,34 L/L). La forte réaction leucémique était composée de neutrophiles, de monocytes et de lymphocytes. Le chien a été diagnostiqué avec une infection hépatique à Staphylococcus pseudointermedius sur la base d'aspirations et de cultures de foie. L'amoxicilline-acide clavulanique a été prescrit. Une échographie abdominale de contrôle et un CBC répété 4 semaines après l'examen initial étaient sans particularité. L'hyperleucocytose néonatale est bien décrite en médecine humaine mais les études vétérinaires chez les nouveau-nés de petits animaux sont rares.Message clinique clé :L'hyperleucocytose chez les chiens adultes peut être causée par une leucémie ou des réactions leucémiques. La septicémie généralisée est l'une des principales causes de réactions leucémiques chez les chiens et les chats adultes. Chez les chiots, la néoplasie est moins probable et d'autres causes doivent être recherchées. Semblables aux nouveaunés humains, les chiots peuvent développer une forte réaction leucémique lors d'une infection, même s'il ne s'agit pas d'un processus septique généralisé.(Traduit par Dr Serge Messier).

Hematology and biochemistry reference intervals for Ontario commercial nursing pigs close to the time of weaning.

The evaluation of pig hematology and biochemistry parameters is rarely done largely due to the costs associated with laboratory testing and labor, and the limited availability of reference intervals needed for interpretation. Within-herd and between-herd biological variation of these values also make it difficult to establish reference intervals. Regardless, baseline reference intervals are important to aid veterinarians in the interpretation of blood parameters for the diagnosis and treatment of diseased swine. The objective of this research was to provide reference intervals for hematology and biochemistry parameters of 3-week-old commercial nursing piglets in Ontario. A total of 1032 pigs lacking clinical signs of disease from 20 swine farms were sampled for hematology and iron panel evaluation, with biochemistry analysis performed on a subset of 189 randomly selected pigs. The 95% reference interval, mean, median, range, and 90% confidence intervals were calculated for each parameter.

Intervalles de références de l'hématologie et de la biochimie pour des porcelets à l'allaitement vers le moment du sevrage dans des élevages commerciaux de l'Ontario. L'évaluation des paramètres hématologiques et biochimiques des porcs est rarement réalisée surtout en raison des coûts associés aux tests de laboratoire ainsi qu'à la main-d'œuvre et à la disponibilité limitée d'intervalles de référence requis pour l'interprétation. La variation de ces valeurs au sein de troupeaux et entre les troupeaux complique l'établissement des intervalles de référence. Néanmoins, des intervalles de référence de base sont importants pour appuyer les vétérinaires dans l'interprétation des paramètres sanguins pour le diagnostic et le traitement des porcs malades. Cette recherche avait pour objectif de fournir des intervalles de référence pour les paramètres hématologiques et biochimiques des porcelets commerciaux à l'allaitement âgés de 3 semaines en Ontario. Des échantillons ont été prélevés pour un total de 1032 porcs ne présentant pas de signes cliniques de maladie provenant de 20 fermes porcines pour une évaluation du profil d'hématologie et du fer et une analyse biochimique a été réalisée pour un sous-groupe de 189 porcs choisis au hasard. L'intervalle de référence de 95 %, la moyenne, la médiane, l'étendue de référence et les intervalles de confiance de 90 % ont été calculés pour chaque paramètre.(Traduit par Isabelle Vallières).

Retro-orbital and disseminated B-cell lymphoma in a yellow-collared macaw (Primolius auricollis).

A yellow-collared macaw was presented with unilateral left exophthalmia. The complete blood cell count and biochemistry revealed a heterophilic leukocytosis and elevation in liver parameters, respectively. A computed tomography scan showed a contrast-enhancing retrobulbar mass and hepatomegaly. Cytology of the liver was consistent with a round cell tumor, most likely lymphoma. The bird died after 2 months of palliative care. Postmortem examination confirmed a retro-orbital and disseminated B-cell lymphoma.

Lymphome B rétro-orbital et disséminé chez un ara à collier jaune(Primolius auricollis). Un ara à collier jaune a été présenté avec de l'exophtalmie unilatérale gauche. La formule sanguine complète et la biochimie ont révélé une leucocytose hétérophile et une élévation des paramètres hépatiques, respectivement. La tomodensitométrie à l'aide d'une injection de milieu de contraste a montré une masse rétrobulbaire et une hépatomégalie. La cytologie du foie était conforme à une tumeur à cellules rondes, le plus probablement un lymphome. L'oiseau est mort après 2 mois de soins palliatifs. L'examen postmortem a confirmé un lymphome B rétro-orbital et disséminé.(Traduit par Isabelle Vallières).

Purification of protein C from canine plasma.

BACKGROUND: In order to characterize the functional properties of canine protein C (CnPC), the zymogen needs to be purified from plasma. The goals of this study were (1) to purify protein C from fresh frozen canine plasma by barium chloride and ammonium sulphate precipitation, followed by immunoaffinity chromatography using a monoclonal mouse antibody against human protein C (HPC4) and (2) to characterize this protein's structure. RESULTS: The purified protein contained three glycosylated forms of a heavy chain (~49 kDa) and a glycosylated light chain (~ 25 kDa). Tandem mass spectra of the peptides obtained following trypsin digestion and liquid chromatography identified this protein to be protein C (vitamin K-dependent protein C precursor, gi|62078422) with 100% probability. Three glycosylation sites (Asn139, Asn202, and Asn350) were identified by detection of peptides containing an N-linked glycosylation consensus sequon with a 3-dalton increase in mass following incubation of the protein with PNGase F in 18O-labeled water. Following incubation with Protac (a specific activator of protein C), the heavy chain showed a slight decrease in molecular size and amidolytic activity measured by a synthetic chromogenic substrate containing an amide bond [H-D-(γ-carbobenzoxyl)-lysyl-prolyl-arginine-paranitroanilide diacetate salt]. The amidolytic activity was increased by ~303-fold in the final protein preparation compared to that in plasma. The purified protein showed concentration-dependent anti-factor V and anti-factor VIII activities in canine plasma in coagulometric factor assays. CONCLUSIONS: These studies showed that CnPC could be purified from plasma using HPC4 and that this protein showed amidolytic and anti-coagulant properties upon activation with Protac.

Determination of clopidogrel effect in cats using point-of-care Plateletworks ADP and shipped samples for PFA-200 analysis in a clinical practice setting.

OBJECTIVES: Clopidogrel is the recommended first-line antithrombotic in cats for a variety of conditions; however, it is ineffective in 15-20% of cats. The determination of clopidogrel effectiveness with platelet function assays has historically been limited to specialty centers; however, recent work has suggested that in-hospital or shipped analyses of samples may be feasible. The aim of the present study was to investigate the utility of an in-house analysis and shipping of blood samples collected in primary practices for the determination of clopidogrel effectiveness. METHODS: Citrated blood samples were collected from cats receiving clopidogrel therapy by veterinarians in clinical practices across Canada, a median of 304.4 km from the reference laboratory (range 8-4425). Samples were analyzed in-house using Plateletworks ADP and shipped for remote analysis using PFA-200 P2Y and COL/ADP cartridges. RESULTS: A total of 30 samples were collected from 25 cats. Of these, the percentage of samples analyzable for the presence or absence of the clopidogrel effect was 86% for Plateletworks ADP, 90% for PFA-200 P2Y and 87% for PFA-200 COL/ADP. There was no significant difference in the number of samples unable to be analyzed by each modality (P = 0.689) due to flow obstruction or other sample characteristics. The prevalence of absence of clopidogrel effectiveness on platelet function assays was 8% with the PFA-200 COL/ADP assay, 25% with the PFA-200 P2Y assay and 30% with the Plateletworks ADP assay. CONCLUSIONS AND RELEVANCE: The results of this study confirm that samples of feline blood can be collected in clinical practices and shipped to a reference laboratory for PFA-200 analysis with a high rate of success, comparable to point-of-care analysis.

Investigation of Platelet Function Analyzer 200 platelet function measurements in healthy cats and cats receiving clopidogrel.

The Platelet Function Analyzer 200 (PFA-200; Siemens) is an in vitro substitute for in vivo bleeding time that is designed to investigate platelet function in a more physiologic manner than traditional aggregometry. The analyzer reports a closure time (CT) as a marker of platelet function, and may also report the calculated platelet function measurement primary hemostasis components, PHC1 and PHC2. These incorporate the measured total volume (TV) of blood aspirated and the initial flow rate (IF). We determined, for the COL/ADP and P2Y cartridges, the median total volume (TVmedian), and RIs for CT, IF, TV, PHC1, and PHC2, and investigated the sensitivity and specificity of those parameters at the determined interpretation thresholds in determination of the clopidogrel effect. Healthy client-owned cats were recruited prospectively to determine RIs for CT, IF, TV, PHC1, and PHC2. Healthy blood-donor cats and cats on clopidogrel therapy were included retrospectively to determine test performance. In 20 healthy cats, RIs for COL/ADP were CT (19.5-87.2 s), IF (199-278 µL/min), TV (199-332 µL), PHC1 (94-106%), and PHC2 (52-148%); and for P2Y, CT (4.2-94.3 s), IF (112-208 µL/min), TV (151-294 µL), PHC1 (35-178%), and PHC2 (90-109%). CVs were calculated for all of these values. Specificity for detection of the clopidogrel effect was calculated from a group of healthy blood donors, and sensitivity for detection of the clopidogrel effect from a group of cats with known clopidogrel effect. Sensitivity and specificity were, for COL/ADP: CT (83.3%, 66.6%), IF (41.4%, 83.3%), TV (83.3%, 100%), PHC1 (100%, 100%) and PHC2 (100%, 83.3%); and for P2Y: CT (100%, 94.4%), IF (30%, 44.4%), TV (100%, 94.4%), PHC1 (100%, 100%), and PHC2 (100%, 97.7%). These PFA-200 values may be beneficial in the determination of platelet function in cats.

Validation of storage and shipping of feline blood samples for analysis on the Platelet Function Analyzer-200 for determining the effect of clopidogrel.

BACKGROUND: The Platelet function analyzer-200 (PFA-200) can determine the effect of clopidogrel in cats, but analysis traditionally must be performed at point-of-care (POC). The ability to ship samples of blood to a laboratory would allow widespread access. OBJECTIVES: We aimed to validate the shipping of blood samples for PFA-200 analysis in cats to determine the effect of clopidogrel. METHODS: Twenty healthy cats and 10 cats receiving clopidogrel were recruited. Blood was collected from cats and aliquoted into two samples, one was analyzed at POC within 2 hours using the PFA-200, and the other was packaged and transported to a location 4 km away, stored, and transported back to the lab for analysis the following day. RESULTS: Median closure times (CTs) with the collagen/adenosine diphosphate (COL/ADP) cartridge in healthy cats were 51.5 seconds (POC) and 78.8 seconds (shipped), which were significantly different (P < 0.001), and for cats on clopidogrel, median CTs were 147.5 seconds (POC) and 190 seconds (shipped), which were not significantly different (P = 0.131). Median CTs with the P2Y cartridge in healthy cats were 50.5 seconds (POC) and 64.9 seconds (shipped), which were significantly different (P = 0.03), and in cats receiving clopidogrel, median CTs were 300 seconds (POC) and 300 seconds (shipped) which were not significantly different (P = 1.000). Reference intervals for CTs differed for COL/ADP at POC (19.8-89.7 seconds) and shipped (50.9-161.6 seconds) and for P2Y at POC (35.5-118.8 seconds) and shipped (35.1-108.9 seconds). Receiver operating characteristics showed similar areas under the curve (AUCROCs) regarding the effect of clopidogrel for COL/ADP at POC (0.994 seconds) and shipped (0.932) and for P2Y at POC (0.904 seconds) and shipped (0.975 seconds). When classifying for the presence of clopidogrel effects, Cohen's Kappa was 0.62 for COL/ADP and 1.00 for P2Y. CONCLUSIONS: Shipping blood samples for PFA analysis are feasible with similar performance to POC analyses for determining the effect of clopidogrel in cats.

Extended sample storage for platelet function testing in healthy dogs.

BACKGROUND: Platelet function testing is important for monitoring the effects of antiplatelet therapy but is not readily used due to time constraints for testing and the need for specialized equipment. OBJECTIVES: This study evaluated the effects of various storage methods on selected platelet function tests to determine if delayed platelet function testing is feasible in canine blood samples. Our hypotheses were that platelet function would not decline during storage and, thus, no differences in test results would be found over time. METHODS: Thirteen healthy dogs were studied. Citrated blood samples were tested with a Platelet Function Analyzer-200 (PFA), which mimics high-shear conditions, using P2Y and CADP cartridges, after being held at room temperature for 2 h and refrigerated for 24 and 48 h. Plateletworks (PW), which measures aggregation based on platelet counting, was performed on an optical hematology analyzer using 10-min-old native samples, citrated samples held at room temperature for 3-4 h and refrigerated for 24 and 48 h, and samples stored in the preservative solution, AGGFix, up to 7 days. RESULTS: PFA closure times increased with storage, especially with the P2Y cartridge. Median aggregation with fresh PW was 94%, and this was maintained at all time points (range of median values 88%-94%). Most samples showed decreased, yet still robust (>70%), aggregation with longer storage. Spontaneous aggregation in citrate was noted in most dogs. AGGFix stabilized platelet aggregates to allow for delayed testing. CONCLUSIONS: Delayed platelet function testing is feasible, but ranges of expected values may differ from tests using fresh samples.

Platelet function analyzer-200 closure curve analysis and assessment of flow-obstructed samples.

BACKGROUND: The Platelet function analyzer-200 can determine the effect of clopidogrel in cats. Flow obstruction is an error that causes uninterpretable results. Closure curves and parameters initial flow rate (IF) and total volume (TV) are displayed by the PFA-200 and may allow interpretation of results in cases of flow obstruction. The primary hemostasis components (PHC) are calculated values that normalize these parameters. OBJECTIVES: To determine if closure curves and research parameters allow detecting the effect of clopidogrel in cases of flow obstruction. METHODS: A review of closure curves identified those with flow obstruction and paired analysis that did not. Non-flow-obstructed curves were used to categorize curves with respect to clopidogrel effects. IF, TV, PHC(1), and PHC(2) were evaluated to determine if these could be used to categorize if a sample exhibited the effects of clopidogrel. Curves were visually analyzed, and characteristics identified that were more common with or without the effect of clopidogrel. Visual analysis of curves was performed by blinded observers to determine if a visual analysis was able to predict the effect of clopidogrel. RESULTS: Analysis of parameters was able to predict closure or non-closure in flow-obstructed curves. TV, PHC(1), and PHC(2) had area under the curve of the receiver operating characteristics of 0.79, 0.79, and 0.87. Visual curve analysis was unable to predict closure, with an average accuracy of only 55%, among three reviewers. Agreement between reviewers was poor (Fleiss' Kappa 0.06). CONCLUSIONS: Visual curve analysis was unable to determine the effect of clopidogrel in flow-obstructed samples. Numerical parameters were able to detect the effect of clopidogrel with a high degree of accuracy in flow-obstructed samples.

Validation of Plateletworks ADP for the ProCyte Dx analyzer.

BACKGROUND: Platelet function testing in cats allows determination of clopidogrel effect. Plateletworks assesses aggregation based on decreasing platelet counts on hematology analyzers in response to agonists. It has not been validated for the IDEXX ProCyte Dx analyzer. Ideal time to perform analysis and the utility of other platelet parameters have not been fully assessed. OBJECTIVES: To validate Plateletworks ADP on the ProCyte Dx, to investigate the utility of various platelet parameters using Plateletworks ADP, and determine the ideal time to perform analysis. ANIMALS: Twenty healthy cats recruited from the general population used for transference of reference intervals to a new analyzer, and 10 cats receiving clopidogrel to determine clopidogrel effect. METHODS: Plateletworks ADP using the ProCyte Dx and ADVIA 2120i analyzer was run simultaneously in both healthy cats and cats receiving clopidogrel, and CBC results at different timepoints were compared between analyzers. RESULTS: Aggregation was significantly different (P < .001) between analyzers. Cohen's kappa showed almost perfect agreement for determination of clopidogrel effect, and the area under the curve of the receiver operating characteristic was 1.0. Lower limits of the aggregation reference interval in healthy cats were 28.8% on the ProCyte Dx and 12.5% on the ADVIA 2120i. Coefficients of variation for platelet parameters were not different between analyzers. No significant changes in mean platelet volume, plateletcrit, large platelets, and mean platelet component were identified. No significant change in aggregation was observed within the first hour after phlebotomy. CONCLUSIONS AND CLINICAL IMPORTANCE: Our study validated the Plateletworks ADP system on the ProCyte Dx analyzer. Samples may be analyzed up to 1 h after collection.

Treatment of myeloid neoplasia with doxorubicin and cytarabine in 11 dogs.

Myelodysplastic syndrome (MDS) and acute myeloid leukaemia (AML) are primary myeloid neoplasms in dogs generally considered to have a poor outcome. In this study, we assessed toxicity, efficacy and outcome of concurrent administration of doxorubicin and cytarabine in 11 dogs with myeloid neoplasia. Bone marrow specimens were reviewed by three pathologists and classified as either MDS (n = 2), high grade MDS/early AML (MDS/AML; n = 4) or AML (n = 5). The median number of treatment cycles was 5 (range 1-9) and resolution of cytopenia was reported in 7 of 11 dogs including 2 dogs with MDS, 2 dogs with MDS/AML, and 3 dogs with AML. The median duration of remission in the seven responders was 344 days (range 109-1428) and the median overall survival for all dogs was 369 days. Adverse events consisted of predominantly low-grade gastrointestinal illness and myelosuppression. Three dogs developed grade V toxicity manifesting with heart failure (n = 2) at 369 and 1170 days after diagnosis and acute gastrointestinal side effects (n =1). Despite a limited sample size, these results suggest that a doxorubicin and cytarabine protocol may be considered as a therapeutic option in dogs with myeloid neoplasia. Protocol safety, in particular regarding myocardial toxicity, and efficacy should be further investigated.

Effects of a 0.3% cholesterol diet and a 20% fat diet on plasma lipids and lipoproteins in Quaker parrots (Myiopsitta monachus).

BACKGROUND: Lipid disorders are common in captive psittacine birds, but associated changes in blood lipids and lipoproteins have not been well characterized. The Quaker parrot is prone to dyslipidemia and has been extensively used as an experimental model. OBJECTIVES: We aimed to study the effects of a 0.3% cholesterol diet and a 20% fat diet on plasma lipids and lipoproteins in Quaker parrots. METHODS: Two crossover studies were performed with each diet. During each study, 12 parrots were divided into two groups fed the treatment or control diet for 2 weeks. After a 2-month wash-out period, the groups were reversed. At the end of each period, plasma lipidomics and lipoprotein profiling were performed. Data were analyzed by univariate tests adjusted for false discovery rates, volcano plots, and enrichment analyses. RESULTS: The cholesterol diet induced changes in many plasma lipids and lipoproteins. Total cholesterol and cholesteryl esters were significantly and markedly elevated. Ceramides were the second subclass of lipids that were elevated. Several glycerophosphocholines, sphingomyelins, and one diacylglycerol were also significantly elevated, albeit to a lesser magnitude. All lipoproteins were elevated, with the greatest increase seen in non-HDL. The fat diet mainly resulted in a decrease in plasma glycerolipids and an increase in acylcarnitines. Lipoprotein plasma levels remained unchanged. CONCLUSIONS: Quaker parrots fed a 0.3% cholesterol diet showed profound and complex dyslipidemic changes that could be used to further study lipid disorders and their management in psittacine birds. A 20% fat diet higher in n-6 polyunsaturated fatty acids did not lead to dyslipidemia.

Validation of a commercial human ELISA to measure hyaluronic acid concentration in feline plasma.

Our goal was to validate a human hyaluronic acid (HA) ELISA (Hyaluronic acid plus ELISA; TECOmedical Group) for use in feline plasma. Plasma from 5 healthy cats and 5 critically ill cats was used for validation of the assay. Validation methods performed included intra- and inter-assay variability, spike-and-recovery, and dilutional linearity. All measurements were performed in duplicate. The precision study revealed good intra-assay CV of 7.4-8.9%; inter-assay CV was 3.4-4.2%. Extraction efficiency via spiking tests yielded mean recovery of 89.6%. The assay met criteria for acceptable linearity using 3 serial dilutions. Our results demonstrate that this commercial HA ELISA had acceptable analytical performance using feline plasma and could be a useful tool in the veterinary clinical research setting.

Cyclic hematopoiesis in a mixed-breed dog: case report and brief review.

An 8-wk-old, male, mixed-breed puppy was adopted from a rescue organization. From the time of adoption, the puppy suffered episodes of illness affecting various organ systems, which resolved with supportive therapy but relapsed once medical therapy was discontinued. Review of the hematologic data revealed cyclic fluctuations in circulating blood cells. Cyclicity was most prominent in neutrophils, with recurrent severe neutropenia. Neutropenic episodes lasted 5-6 d, with regular cycles of 11-14 d between nadir neutrophil counts. Genetic testing determined that the patient was homozygous mutant for the frameshift mutation in the adaptor protein complex 3 ß-subunit (AP3B1) gene, originally identified in gray collies with cyclic hematopoiesis (CH). Pedigree information was not available, but the patient's features were phenotypically distinct from those of collies. We describe here a case of the AP3B1 mutation in a mixed-breed dog that did not resemble a collie, undescribed previously, to our knowledge. Our findings indicate that the AP3B1 mutation and CH are present within the general canine population and are not restricted to collies.

Evaluation of lymphocyte-specific programmed cell death protein 1 receptor expression and cytokines in blood and urine in canine urothelial carcinoma patients.

Urothelial carcinoma (UC) is the most common urinary tumour in dogs. Despite a range of treatment options, prognosis remains poor in dogs. In people, breakthroughs with checkpoint inhibitors have established new standards of care for muscle-invasive bladder cancer patients and elevated levels of programmed cell death protein 1 (PD-1) suggest immune checkpoint blockade may be a novel target for therapy. The goal of this study was to determine if canine UC patients express elevated levels of lymphocyte-specific PD-1 and/or urinary cytokine biomarkers compared to healthy dogs. Paired blood and urine were evaluated in 10 canine UC patients, five cystitis patients and 10 control dogs for lymphocyte-specific PD-1 expression via flow cytometry and relative cytokine expression. In UC patients, PD-1 expression was significantly elevated on CD8+ lymphocytes in urine samples. UC patients had a higher CD4:CD8 ratio in their urine compared to healthy dogs, however, there was no significant variation in the CD8:Treg ratio between any group. Cystitis patients had significantly elevated levels of CD4+ T cells, CD8+ T cells and Tregs in their blood samples compared to UC patients and healthy dogs. Cytokine analysis demonstrated significant elevations in urinary cytokines (granulocyte-macrophage colony-stimulating factor, interferon-gamma [IFN-γ], interleukin (IL)-2, IL-6 IL-7, IL-8 and IL-15, IP-10, KC-like, IL-18, monocyte chemoattractant protein-1 and tumour necrosis factor-alpha). Several of these cytokines have been previously correlated with both lymphocyte-specific PD-1 expression (IFN-γ, IL-2, IL-7 and IL-15) in muscle-invasive urothelial carcinoma in humans. Our results provide evidence of urinary lymphocyte PD-1 expression and future studies could elucidate whether veterinary UC patients will respond favourably to anti-PD-1 immune checkpoint inhibitor therapy.

Evaluation of PD-1 and PD-L1 expression in canine urothelial carcinoma cell lines.

Urothelial carcinoma (UC) is the most common urinary tumor in dogs and despite combinational therapies, only modest improvements in survival have been achieved in recent years. Given the utility of monoclonal antibodies against PD-1 and PD-L1 in human UC, we evaluated the protein and mRNA expression in three established canine urothelial carcinoma cell lines. Flow cytometry and western blot analysis confirmed cell line expression of both molecules in varying degrees. Reverse transcription PCR (RT-PCR) documented mRNA expression in all three cell lines for both PD-1 and PD-L1. Fluorescence microscopy was consistent with strong PD-1 and PD-L1 expression in the canine cell lines and was in line with previous human literature. Importantly, the flow cytometry work described in this study revealed higher cell intrinsic PD-1 expression in these cell lines which may have implications for tumor behavior and potential treatment opportunities in the future. Further work is necessary to determine the expression patterns in canine UC and potential for benefit with immunotherapy directed against PD-1 and PD-L1.

Use of serum hyaluronic acid as a biomarker of endothelial glycocalyx degradation in dogs with septic peritonitis.

OBJECTIVE: To describe daily changes in serum concentrations of hyaluronic acid (HA), a biomarker of endothelial glycocalyx degradation, in dogs with septic peritonitis and to determine whether relationships exist among serum concentrations of HA and biomarkers of inflammation and patient fluid status. ANIMALS: 8 client-owned dogs. PROCEDURES: Serum samples that had been collected for a previous study and stored at -80°C were used. Blood samples were collected at admission and daily thereafter during hospitalization and were analyzed for concentrations of HA and interleukins 6, 8, and 10. Patient data including acute patient physiologic and laboratory evaluation score, type and amount of fluids administered daily, and daily CBC and lactate concentration results were recorded. To determine the significant predictors of HA concentration, a general linear mixed model for repeated measures was developed. RESULTS: All dogs survived to discharge. Concentrations of HA ranged from 18 to 1,050 ng/mL (interquartile [25th to 75th percentile] range, 49 to 119 ng/mL) throughout hospitalization. Interleukin-6 concentration was a significant predictor of HA concentration as was total administered daily fluid volume when accounting for interleukin-6 concentration. When fluid volume was analyzed independent of inflammatory status, fluid volume was not a significant predictor. Concentrations of HA did not significantly change over time but tended to increase on day 2 or 3 of hospitalization. CONCLUSIONS AND CLINICAL RELEVANCE: Results supported the theory that inflammation is associated with endothelial glycocalyx degradation. Dogs recovering from septic peritonitis may become more susceptible to further endothelial glycocalyx damage as increasing fluid volumes are administered.

Serum interleukin 17 concentrations in dogs with immune-mediated hemolytic anemia.

BACKGROUND: Increased serum interleukin 17 (IL-17) concentration has been associated with the immunopathogenesis of autoimmune hemolytic anemia in humans. No data are available about IL-17 in immune-mediated hemolytic anemia (IMHA) of dogs. OBJECTIVES: Monitor changes in serum IL-17 concentration during the acute stages of IMHA in dogs, compared with results in healthy dogs, and its relationship with outcome. ANIMALS: Thirty-one client-owned dogs with primary IMHA and 27 healthy dogs. METHODS: Quantification of serum IL-17 concentration using a commercially available ELISA kit at the time of admission (D0), after 48 hours (D2) and after 96 hours (D4) as compared to concentration in healthy dogs. The IMHA dogs were classified as survivors if discharged from hospital, or nonsurvivors for any cause of in-hospital mortality. RESULTS: Mean serum IL-17 concentration was higher in dogs with IMHA on admission compared with healthy dogs (D0), but this difference was not significant (mean, 19.52 pg/mL vs 10.52 pg/mL, respectively, P = .17). Throughout hospitalization, serum IL-17 concentration significantly decreased in survivors. Serum IL-17 concentration at D0 was not different between survivors and nonsurvivors, but surviving dogs had significantly lower serum IL-17 concentration at D2 and D4 (P = .04 and P = .004, respectively) compared with nonsurviving dogs. No correlation was found between serum IL-17 concentration and serum total bilirubin or lactate concentrations or CBC parameters. CONCLUSION AND CLINICAL IMPORTANCE: Serum IL-17 concentration remained significantly higher in nonsurviving IMHA dogs whereas it significantly decreased during hospitalization in survivors, making serum IL-17 concentration a potential biomarker for severity and response to treatment in IMHA.

Effects of aspirin, carprofen, deracoxib, and meloxicam on platelet function and systemic prostaglandin concentrations in healthy dogs.

OBJECTIVE: To determine effects of therapeutic dosages of aspirin, carprofen, deracoxib, and meloxicam on platelet function and systemic prostaglandin concentrations in healthy dogs. ANIMALS: 10 hound-crossbred dogs. PROCEDURES: Aspirin (10 mg/kg, PO, q 12 h), carprofen (4.4 mg/kg, PO, q 24 h), deracoxib (2 mg/kg, PO, q 24 h), meloxicam (0.1 mg/kg, PO, q 24 h), and a placebo were administered for 7 days in a random order to each of 10 healthy dogs; there was a 21-day washout period between subsequent treatments. One-stage prothrombin time (PT), activated partial thromboplastin time (aPTT), fibrinogen concentration, and plasma concentrations of thromboxane (TX)B(2) and 6-keto prostaglandin (PG)F(1alpha) were measured before and after treatment administration. Platelet function was assessed by use of a platelet-function analyzer and aggregation. RESULTS: Aspirin, carprofen, and meloxicam did not significantly affect platelet function. Deracoxib caused a mild decrease in platelet aggregation induced by 50microM ADP. Platelet number, Hct, PT, aPTT, and plasma TXB(2) and 6-keto PGF(1alpha) concentrations were unchanged after NSAID administration. Meloxicam administration resulted in a significant decrease in fibrinogen concentration, but results remained within the laboratory reference interval. CONCLUSIONS AND CLINICAL RELEVANCE: Oral administration of commonly used NSAIDs at therapeutic dosages in healthy dogs did not alter plasma TXB(2) and 6-keto PGF(1alpha) concentrations. Deracoxib administration resulted in a minor abnormality in platelet aggregation. Anti-inflammatory doses of aspirin did not affect platelet function as measured by use of optical aggregometry and a platelet-function analyzer. Further evaluation of the effects of aspirin and cyclooxygenase-2-selective inhibitors on hemostasis should be performed.