Demographic factors associated with joint supplement use in dogs from the Dog Aging Project

Osteoarthritis (OA) is one of the most prevalent age-related chronic conditions that afflict companion dogs, and multiple joint supplements are available to prevent or treat OA, though the efficacy of these treatments is controversial. While the demographic factors that are associated with OA diagnosis are well established, the factors that are associated with joint supplement use are not as well studied. Using data collected from the Dog Aging Project, we analyzed owner survey responses regarding joint supplement administration and OA diagnosis for 26,951 adult dogs. In this cross-sectional analysis, logistic regression models and odds-ratios (OR) were employed to determine demographic factors of dogs and their owners that were associated with joint supplement administration. Forty percent of adult dogs in our population were given some type of joint supplement. Perhaps not surprisingly, dogs of older age, larger size, and those that were ever overweight were more likely to receive a joint supplement. Younger owner age, urban living, owner education, and feeding commercial dry food were associated with a reduced likelihood of administration of joint supplements to dogs. Interestingly, mixed breed dogs were also less likely to be administered a joint supplement (OR: 0.73). Dogs with a clinical diagnosis of OA were more likely to receive a joint supplement than those without a reported OA diagnosis (OR: 3.82). Neutered dogs were more likely to have a diagnosis of OA, even after controlling for other demographic factors, yet their prevalence of joint supplement administration was the same as intact dogs. Overall, joint supplement use appears to be high in our large population of dogs in the United States. Prospective studies are needed to determine if joint supplements are more commonly administered as a preventative for OA or after an OA clinical diagnosis

Silicone tags as an effective method of monitoring environmental contaminant exposures in a geographically diverse sample of dogs from the Dog Aging Project

IntroductionCompanion animals offer a unique opportunity to investigate risk factors and exposures in our shared environment. Passive sampling techniques have proven effective in capturing environmental exposures in dogs and humans.MethodsIn a pilot study, we deployed silicone monitoring devices (tags) on the collars of a sample of 15 dogs from the Dog Aging Project Pack cohort for a period of 120 h (5 days). We extracted and analyzed the tags via gas chromatography–mass spectrometry for 119 chemical compounds in and around participants’ homes.ResultsAnalytes belonging to the following chemical classes were detected: brominated flame retardants (BFRs), organophosphate esters (OPEs), polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), pesticides, phthalates, and personal care products. The types and amounts of analytes detected varied substantially among participants.DiscussionData from this pilot study indicate that silicone dog tags are an effective means to detect and measure chemical exposure in and around pet dogs’ households. Having created a sound methodological infrastructure, we will deploy tags to a geographically diverse and larger sample size of Dog Aging Project participants with a goal of further assessing geographic variation in exposures

Decreased antithrombin activity and inflammation in cats

Objectives The objective of this study was to determine if inflammatory markers are associated with antithrombin activity in cats. Methods For a retrospective population of 231 cats admitted to a referral hospital, antithrombin activity was classified as decreased (n = 77), intermediate (n = 97) or in the upper quartile (n = 57). Odds ratios (ORs) were calculated for an association between decreased or upper quartile activity and hypoalbuminemia, hyperfibrinogenemia, band neutrophilia and toxic change. Multiple logistic regression was performed to determine if an association between band neutrophilia and decreased antithrombin activity was independent of decreased hepatic synthesis, consumptive coagulopathy or protein loss. Results Cats with decreased antithrombin activity were more likely than cats with intermediate-to-upper quartile activity to have band neutrophilia (OR 2.85, P = 0.0050), hypoalbuminemia (OR 12.1, P &lt;0.0001) or toxic neutrophils (OR 4.47, P &lt;0.0001). Cats with antithrombin activity in the upper quartile were less likely than those with intermediate-to-low activity to have hypoalbuminemia (OR 0.31, P = 0.0023) or toxic neutrophils (OR 0.44, P = 0.033). In a regression model that included other mechanisms for decreased antithrombin, band neutrophilia remained associated with decreased antithrombin activity (adjusted OR 2.62, P = 0.013). Conclusions and relevance Contrary to previous studies suggesting antithrombin is a feline positive acute phase protein, this study demonstrates an association between decreased antithrombin activity and inflammation. Further studies are needed to determine the mechanistic basis of this association. </jats:sec

Canine Neutrophil Extracellular Traps Enhance Clot Formation and Delay Lysis

Autoimmune diseases increase the risk of thrombosis. Neutrophil extracellular traps (NETs) are webs of DNA and protein that may mediate thrombosis in autoimmune diseases. Human and murine studies show NET-releasing neutrophils within a thrombus promote its growth, but it is unclear to what extent NET fragments released into circulation during inflammation are prothrombotic. This study hypothesized that canine NETs promote clot formation and impair lysis even in the absence of neutrophils. NETs were prepared from PMA-stimulated neutrophils and added to fibrinogen and thrombin or to recalcified pooled canine platelet-poor plasma, tissue factor, and tissue plasminogen activator. Clot formation and lysis were measured spectrophotometrically. NETs did not alter fibrin clot formation, but NETs increased maximum clot formation velocity ( P = .001) and delayed lysis ( P = .009) of plasma clots compared with supernatants from nonstimulated neutrophils. DNase digestion of NETs reduced their effect on clot lysis but not maximum clot formation velocity. This suggested impaired lysis was principally mediated by DNA within NETs but that NET proteins were principally responsible for increased speed of clot formation. Previous reports suggested elastase or histones might be responsible for the effect of NETs on clot formation. Elastase activity was greatly reduced by plasma, and addition of histones to plasma did not increase formation velocity, suggesting these proteins were not responsible for increasing maximum formation velocity. This study showed that NETs enhanced clot formation and impaired clot lysis in canine platelet-poor plasma. These in vitro findings suggest both NET proteins and DNA may contribute to thrombosis in inflammatory disease. </jats:p

Canine Neutrophil Extracellular Traps Have Pro-Coagulant and Anti-Fibrinolytic Effects

Abstract Pet dogs experience similar environmental influences to their owners, and thus provide valuable spontaneous models for investigation of novel therapies for human disease. DNase and treatments targeting neutrophil extracellular trap (NET) proteins show promise in murine thrombosis models. Dogs at risk of thrombosis could provide evidence for the efficacy of these treatments in a second species, but the pro-thrombotic effects of NETs on canine plasma must be confirmed. We isolated neutrophils from healthy canine blood by density gradient and stimulated NET release using phorbol 12-myristate 13-acetate (PMA). To release NETs into suspension, culture media was replaced by the restriction enzyme Alu1. Supernatants were centrifuged to remove any intact cells. By gel electrophoresis, NET fragments were predominantly 600-1000 base pairs which is sufficient to simultaneously bind plasmin and fibrin (Arterioscler Thromb Vasc Biol., 2015, 35: 2544-53). To distinguish the effects of DNA and NET proteins, a subset of NET fragments were further digested with DNase. Supernatants were identically prepared from non-stimulated neutrophils. DNA concentration was 106ng/ml (sd 49, n=5 dogs) for non-stimulated supernatants; 401ng/ml (sd 94) for NET supernatants and reduced to 113ng/ml (sd 18) by DNase. The effect of NETs on clot formation and lysis were measured spectrophotometrically. To bring fibrinogen into the range typically found in dogs suffering from pro-thrombotic disease, 4.2mg/ml of plasminogen-depleted fibrinogen was mixed with pooled platelet poor plasma from healthy dogs. Plasma was incubated with non-stimulated supernatants; NETs +/- DNase or controls for effects of restriction enzymes, PMA and culture media. We added tissue factor, 5mM calcium chloride and 100µg/ml tissue plasminogen activator and measured absorption at 405nm q. 10 seconds for 25 minutes. NETs increased maximum formation velocity (MFV) (192mOD/min sd. 12, p=0.0011), measured by a line of best fit for the upswing of the curve, and delayed time to 50% clot lysis (448s sd. 43) compared with non-stimulated supernatants (165mOD/min sd. 12; 395s sd 38; n=5). Using a repeated measures ANOVA followed by paired t-tests with Tukey's correction, the effect on MFV (p=0.0089) but not time to 50% clot lysis (p=0.08) remained significant after DNase digestion. NETs did not have detectable effects on area under the curve (AUC) or peak (pANOVA &gt;0.2) (Fig. 1). Reagent controls had no detectable effects on any parameter (pANOVA≥0.717; 5-6 technical replicates). Reduction in fibrinolysis by DNA replicates its effect on human plasma (Arterioscler Thromb Vasc Biol., 2015, 35: 2544-53). The lack of effect of DNA on canine clot formation was surprising given the ability of DNA to increase human plasma thrombin generation (Arterioscler Thromb Vasc Biol. 2014, 34:1977-84). To determine if this was a species difference or a concentration dependent effect, DNA from canine leukocytes was added to plasma at a final concentration of 200ng/ml (approximately equal to the final concentration in our NET studies) or 1000ng/ml. At 1000ng/ml but not 200ng/ml, DNA increased MFV (p=0.038). To determine if NET proteins were increasing MFV by a direct effect on fibrinogen or thrombin, we next studied effects of NETs on thrombin induced clotting of plasminogen-depleted fibrinogen. NETs with or without DNase digestion had no detectable effects on peak, AUC, MFV or fibrin fiber diameter estimated by turbidity. This suggests NET proteins increase plasma clot formation rate through interactions with coagulation components other than thrombin or fibrinogen. Based on human and murine studies showing NET proteins inhibit endogenous anticoagulants, we selected histones and elastase as candidate proteins for further investigation. Purified histones from calf thymus added to plasma at approximately the concentration present in NET supernatants (150ng/ml) had no effect on MFV. Pre-incubation of elastase with canine plasma abolished elastase activity measured by N-Succinyl-Ala-Ala-Ala-p-nitroanilide. Therefore, it is unlikely that the ability of canine NET proteins to increase MFV is mediated by elastase or histones alone. In conclusion, NETs increase clot formation rate and delay lysis in canine platelet poor plasma. Since effects were mediated by both proteins and DNA, dogs are a promising model for trials of both DNase and therapies targeting NET proteins. Disclosures No relevant conflicts of interest to declare. </jats:sec

Positive predictive value of albumin: globulin ratio for feline infectious peritonitis in a mid-western referral hospital population

Low albumin to globulin ratio has been found previously to have a high positive predictive value for feline infectious peritonitis (FIP) in cats with clinical signs highly suggestive of the disease. However, FIP can have a more vague clinical presentation. This retrospective study found that the positive predictive value of an albumin:globulin (A:G) ratio of &lt;0.8 and &lt;0.6 was only 12.5% and 25%, respectively, in a group of 100 cats with one or more clinical signs consistent with FIP. The negative predictive value was 100% and 99% for an A:G ratio of &lt;0.8 and A:G&lt;0.6%, respectively. Therefore, when the prevalence of FIP is low, the A:G ratio is useful to rule out FIP but is not helpful in making a positive diagnosis of FIP. </jats:p