Persistent hypercoagulability in dogs envenomated by the European adder (Vipera berus berus).

BACKGROUND: Envenomation by the European adder, Vipera berus berus (Vbb), is a medical emergency. The overall in vivo haemostatic effects of pro- and anticoagulant components in Vbb venom, and the downstream effects of cellular injury and systemic inflammation, are unclear. OBJECTIVES: To longitudinally describe the global coagulation status of dogs after Vbb envenomation and compare to healthy controls. A secondary aim was to investigate differences between dogs treated with and without antivenom. METHODS: Citrated plasma was collected at presentation, 12 hours (h), 24 h, 36 h and 15 days after bite from 28 dogs envenomated by Vbb, and from 28 healthy controls at a single timepoint. Thrombin generation (initiated with and without exogenous phospholipids and tissue factor), thrombin-antithrombin (TAT)-complexes and the procoagulant activity of phosphatidylserine (PS)-expressing extracellular vesicles (EVs), expressed as PS-equivalents, were measured. RESULTS: At presentation the envenomated dogs were hypercoagulable compared to controls, measured as increased thrombin generation, TAT-complexes and PS-equivalents. The hypercoagulability decreased gradually but compared to controls thrombin generation and PS-equivalents were still increased at day 15. The discrepancy in peak thrombin between envenomated dogs and controls was greater when the measurement was phospholipid-dependent, indicating that PS-positive EVs contribute to hypercoagulability. Lag time was shorter in non-antivenom treated dogs, compared to antivenom treated dogs <24 h after envenomation. CONCLUSIONS: Hypercoagulability was measured in dogs up to 15 days after Vbb envenomation. Dogs treated with antivenom may be less hypercoagulable than their non-antivenom treated counterparts. Thrombin generation is a promising diagnostic and monitoring tool for Vbb envenomation.

Evaluation of Urinary Clusterin and Cystatin B as Biomarkers for Renal Injury in Dogs Envenomated by the European Adder (Vipera berus).

Dogs are commonly bitten by the European adder (Vipera berus) but studies investigating the effects of envenomation are limited. Snakebite-related kidney injury is reported in dogs but diagnosis of acute kidney injury (AKI) might be limited by the insensitivity of routinely used renal function biomarkers. The aim of this study was to evaluate novel biomarkers of renal injury (urinary cystatin B and urinary clusterin) and biomarkers of renal function (serum creatinine and serum symmetric dimethylarginine), and urine protein to creatinine ratio in dogs envenomated by V. berus. Biomarkers were measured at presentation (T1), 12 hours (T2), 24 hours (T3), 36 hours (T4), and 14 days (T5) after snakebite and compared to a group of healthy control dogs. A secondary aim was to investigate the association between biomarker concentrations and severity of clinical signs of envenomation using a snakebite severity score (SSS). Urinary cystatin B concentrations were significantly higher at all timepoints in envenomated dogs compared to controls (P < .010), except for T5 (P = .222). Absolute urinary clusterin concentrations were not significantly different to controls at any timepoint. Compared to controls, serum creatinine and serum symmetric dimethylarginine concentrations were significantly lower in envenomated dogs at T1-T4 (P < .036) and T2-T4 (P < .036), respectively. Urine protein to creatinine ratio was higher in envenomated dogs compared to controls at T2 and T3. Urinary cystatin B concentrations at T1 were correlated with SSS (Spearman's ρ = 0.690, P < .001). The increased urinary cystatin B concentrations observed in dogs envenomated by V. berus in comparison to controls may indicate renal tubular injury in these patients.

Venom-Induced Blood Disturbances by Palearctic Viperid Snakes, and Their Relative Neutralization by Antivenoms and Enzyme-Inhibitors.

Palearctic vipers are medically significant snakes in the genera Daboia, Macrovipera, Montivipera, and Vipera which occur throughout Europe, Central Asia, Near and Middle East. While the ancestral condition is that of a small-bodied, lowland species, extensive diversification has occurred in body size, and niche specialization. Using 27 venom samples and a panel of in vitro coagulation assays, we evaluated the relative coagulotoxic potency of Palearctic viper venoms and compared their neutralization by three antivenoms (Insoserp Europe, VIPERFAV and ViperaTAb) and two metalloprotease inhibitors (prinomastat and DMPS). We show that variation in morphology parallels variation in the Factor X activating procoagulant toxicity, with the three convergent evolutions of larger body sizes (Daboia genus, Macrovipera genus, and Vipera ammodytes uniquely within the Vipera genus) were each accompanied by a significant increase in procoagulant potency. In contrast, the two convergent evolutions of high altitude specialization (the Montivipera genus and Vipera latastei uniquely within the Vipera genus) were each accompanied by a shift away from procoagulant action, with the Montivipera species being particularly potently anticoagulant. Inoserp Europe and VIPERFAV antivenoms were both effective against a broad range of Vipera species, with Inoserp able to neutralize additional species relative to VIPERFAV, reflective of its more complex antivenom immunization mixture. In contrast, ViperaTAb was extremely potent in neutralizing V. berus but, reflective of this being a monovalent antivenom, it was not effective against other Vipera species. The enzyme inhibitor prinomastat efficiently neutralized the metalloprotease-driven Factor X activation of the procoagulant venoms. In contrast, DMPS (2,3-dimercapto-1-propanesulfonic acid), which as been suggested as another potential treatment option in the absence of antivenom, DMPS failed against all venoms tested. Overall, our results highlight the evolutionary variations within Palearctic vipers and help to inform clinical management of viper envenomation.

Serial serum creatinine, SDMA and urinary acute kidney injury biomarker measurements in dogs envenomated by the European adder (Vipera berus).

BACKGROUND: Acute kidney injury (AKI) is associated with high morbidity and mortality in dogs, but diagnosis may be impaired due the insensitivity of routine renal function biomarkers to detect earlier or milder forms of injury. Snake envenomation is one of several causes of AKI in dogs and humans. Dogs are commonly envenomated by the European adder (Vipera berus) between April and October each year, but few studies exist examining serial serum creatinine (sCr) and symmetric dimethylarginine (SDMA) measurements and AKI biomarkers in these dogs. Novel urinary biomarkers could improve clinical outcome by allowing earlier diagnosis of and intervention in AKI. The aim of this study was to assess the presence of AKI in dogs envenomated by V. berus at 12, 24 and 36 h after bite, as well as 14 days later, using sCr, SDMA and a panel of urinary AKI biomarkers normalised to urine creatinine (uCr), compared to a group of healthy control dogs. RESULTS: Thirty-five envenomated dogs and 35 control dogs were included. Serum creatinine did not exceed the upper reference limit at any time point in any dog after envenomation. Serum SDMA did not exceed 0.89 µmol/L in any dog. Compared to controls, urinary albumin/uCr, neutrophil gelatinase-associated lipocalin/uCr and monocyte chemotactic protein-1/uCr were significantly elevated 12 h (P <  0.0001, P <  0.0001, P = 0.01), 24 h (P <  0.001, P <  0.001, P = 0.002) and 36 h (P <  0.001, P <  0.001, P = 0.0008) after bite. Osteopontin/uCr was higher 24 and 36 h after bite (P < 0.0001), kidney injury molecule-1/uCr, interleukin-8/uCr and γ- glutamyl transferase/uCr were significantly higher 36 h after bite (P = 0.003, P = 0.0005, P = 0.001). Urinary cystatin C/uCr was not significantly different to controls at any timepoint. Biomarker/uCr ratios were not significantly different 14 days after envenomation compared to controls. CONCLUSION: Urinary biomarker/Cr ratios are indicative of mild transient, non-azotaemic AKI in dogs envenomated by V. berus.

Ambulatory electrocardiography and serum cardiac troponin I measurement in 21 dogs envenomated by the European adder (Vipera berus).

BACKGROUND: Envenomation by the European adder (Vipera berus) is common in dogs in Europe. Cardiac arrhythmias occur but clinical studies of envenomated dogs are limited. OBJECTIVES: To describe arrhythmias in dogs within 48 hours of envenomation, and investigate associations between arrhythmia grade, serum troponin I (cTnI), and snakebite severity score (SS score). ANIMALS: Twenty-one client-owned dogs bitten by V berus. METHODS: Prospective cohort study of envenomated dogs. Ambulatory electrocardiograms were recorded from presentation to 48 hours after snakebite, and arrhythmias graded 0 to 3 based on frequency and severity. Serum cTnI was measured at presentation, 12 hours, 24 hours, 36 hours, and 14 days after bite. An SS score of 1 to 3 was recorded at admission and based on clinical examination. RESULTS: All dogs survived. Twelve dogs (57%) developed arrhythmias, all of which were ventricular in origin. Severe complex ventricular arrhythmias (VAs) were observed in 6 dogs (29%). Eighty-one percent of dogs (n = 17) had increased cTnI concentrations at 1 or more time points. Dogs that developed arrhythmias had significantly higher concentrations of cTnI at 12 hours (1.67 [0.04-32.68] versus 0.03 [0.01-0.052]; P = .002), 24 hours (1.88 [0.2-14.23] versus 0.06 [0.01-2.06]; P = .009), and 36 hours (3.7 [0.02-16.62] versus 0.06 [0.01-1.33]; P = .006) after bite compared to those that did not. Contingency table analysis showed that SS score was not significantly associated with arrhythmia grade (P = .9). CONCLUSIONS AND CLINICAL IMPORTANCE: Myocardial cell injury, reflected by increased cTnI concentrations and VAs, is common after V berus envenomation in dogs. Prolonged electrocardiography monitoring is advised, particularly where cTnI is increased.

The effect of valproate and levetiracetam on steroidogenesis in forskolin-stimulated H295R cells.

PURPOSE: Endocrine disruptive effects have been frequently observed in patients using antiepileptic drugs (AEDs). Two different AEDs, valproate (VPA) and levetiracetam (LEV), were tested in forskolin-stimulated human adrenal carcinoma (H295R) cells to explore their effect on steroidogenesis. VPA has a long history as an anticonvulsant and is linked with many of the endocrine disorders associated with AED use. LEV is a newer AED, and no endocrine disruptive effects have been reported in humans to date. METHODS: H295R cells, which are capable of full steroidogenesis, were stimulated with forskolin and exposed to either VPA or LEV for 48 h. Medium was collected and analyzed for hormone production. For the VPA-exposed cells, steroidogenic gene expression analysis was also conducted. RESULTS: VPA exposure resulted in a significant reduction in progesterone and estradiol (E2) production, whereas testosterone (T) levels remained unchanged. There were also significant alterations in expression level for most genes analyzed. LEV exposure resulted in a minor, but statistically significant, reduction in T and E2 production. DISCUSSION: Exposure of forskolin-stimulated H295R cells to VPA led to an increased T/E2 ratio through a significant decrease in estradiol production. Gene analysis suggested that VPA affects NR0B1 expression. NR0B1 inhibits promoters of other genes involved in steroidogenesis, and the altered expression of NR0B1 might explain the observed down-regulation in hormone production. The effects of LEV exposure on hormone secretion were not considered to be biologically significant.