A longitudinal study of the blood and urine metabolome of Vipera berus envenomated dogs.

Envenomation of dogs by the common European adder (Vipera berus) is associated with high morbidity. The cytotoxic venom of Vipera berus contains enzymes with the potential to cause acute kidney injury, among other insults, however robust biomarkers for such effects are lacking. A prospective observational follow-up study of naturally envenomated dogs and controls was conducted to fill knowledge gaps regarding canine Vipera berus envenomation, attempt to identify novel biomarkers of envenomation and related kidney injury, and elucidate potential long-term effects. Blood and urine samples were analyzed with a global metabolomics approach using liquid chromatography-mass spectrometry, uncovering numerous features significantly different between cases and controls. After data processing and feature annotation, eight features in blood and 24 features in urine were investigated in order to elucidate their biological relevance. Several of these are associated with AKI, while some may also originate from disturbed fatty acid ß-oxidation and soft tissue damage. A metabolite found in both blood and a venom reference sample may represent identification of a venom component in case dogs. Our findings suggest that envenomated dogs treated according to current best practice are unlikely to suffer permanent injury.

Serum and urinary biomarkers for early detection of acute kidney injury following Hypnale spp. envenoming.

BACKGROUND: Hump-nosed pit viper (HNV; Hypnale spp.) bites account for most venomous snakebites in Sri Lanka. Acute kidney injury (AKI) is the most serious systemic manifestation (1-10%) following HNV envenoming. We aimed to identify the value of functional and injury biomarkers in predicting the development of AKI early following HNV bites. METHODS: We conducted a prospective cohort study of patients with confirmed HNV envenoming presenting to two large tertiary care hospitals in Sri Lanka. Demographics, bite details, clinical effects, complications and treatment data were collected prospectively. Blood and urine samples were collected from patients for coagulation and renal biomarker assays on admission, at 0-4h, 4-8h, 8-16h and 16-24h post-bite and daily until discharge. Follow-up samples were obtained 1 and 3 months post-discharge. Creatinine (sCr) and Cystatin C (sCysC) were measured in serum and kidney injury molecule-1 (uKIM-1), clusterin (uClu), albumin (uAlb), ß2-microglobulin (uß2M), cystatin C (uCysC), neutrophil gelatinase associated lipocalin (uNGAL), osteopontin (uOPN) and trefoil factor-3 (uTFF-3) were measured in urine. Definite HNV bites were based on serum venom specific enzyme immunoassay. Kidney Disease: Improving Global Outcomes (KDIGO) criteria were used to stage AKI. Two patients had chronic kidney disease at 3 month follow-up, both with pre-existing abnormal sCr, and one developed AKI following HNV envenoming. RESULTS: There were 52 patients with confirmed HNV envenoming; median age 48y (Interquartile range [IQR]:40-59y) and 29 (56%) were male. Median time to admission was 1.87h (IQR:1-2.75h). Twelve patients (23%) developed AKI (AKI stage 1 = 7, AKI stage 2 = 1, AKI stage 3 = 4). Levels of five novel biomarkers, the functional marker serum Cystatin C and the damage markers urinary NGAL, cystatin C, ß2-microglobulin and clusterin, were elevated in patients who developed moderate/severe acute kidney injury. sCysC performed the best at 0-4 h post-bite in predicting moderate to severe AKI (AUC-ROC 0.95;95%CI:0.85-1.0) and no biomarker performed better than sCr at later time points. CONCLUSIONS: sCysC appears to be a better marker than sCr for early prediction of moderate to severe AKI following HNV envenoming.

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.

Urinary clusterin and cystatin B as biomarkers of tubular injury in dogs following envenomation by the European adder.

Diagnosing acute kidney injury remains a challenge since the established renal biomarkers, serum creatinine (sCr) and symmetric dimethylarginine (SDMA) reflect glomerular function and not tubular injury. Sensitive tubular markers such as urinary clusterin (uClust) and cystatin B (uCysB) have been proposed to detect AKI at an earlier stage. Since envenomation by the European adder (Vipera berus berus) could serve as a spontaneous disease model of AKI we investigated these new biomarkers in affected dogs. Concentrations of uClust and uCysB as well as sCr and SDMA were analyzed retrospectively in stored samples from 26 dogs with snake envenomation and 13 healthy controls. Higher concentrations of uClust (P < 0.012) and uCysB (P < 0.001) were observed in the snake-envenomed group. Normalization of uClust and uCysB to urinary creatinine did not alter the results. No differences were observed in sCr and SDMA between the snake-envenomed group and the healthy control group. Spearman rank correlation analysis revealed a strong association of uClust with uCysB in the snake-envenomed dogs (r = 0.75 P < 0.001) but not in the healthy controls. The high percentage of snake-envenomed dogs with increased uClust and uCysB concentrations in the absence of increased sCr and SDMA suggests renal tubular injury in the affected dogs. Larger prospective case-controlled studies are warranted to evaluate the clinical utility and prognostic value of these biomarkers.

Transient Megaesophagus Following Coral Snake Envenomation in Three Dogs (2013-2018).

A 12 yr old dachshund, a 7 yr old English springer spaniel, and a 1.5 yr old French bulldog presented following envenomation by a coral snake. Each patient displayed evidence of varying degrees of lower motor neuron dysfunction, but all three developed transient megaesophagus. Two patients developed secondary aspiration pneumonia, with one requiring mechanical ventilation, which the owners declined, resulting in euthanasia. The third developed hypoventilation without aspiration pneumonia, was mechanically ventilated, and was successfully weaned. In the two surviving patients, the megaesophagus resolved by time of discharge. Coral snake envenomation is an uncommon occurrence, and these are the first documented cases of transient megaesophagus secondary to a North American species.

Low accuracy of microscopic hematuria in detecting coagulopathy from Bothrops pit viper bites, Brazilian Amazon.

Introduction: The common lancehead snakes (Bothrops atrox) are responsible for up to 90% of snakebites in the Amazon, especially in remote areas. The prevalence of microhematuria is similar to that of coagulopathy in B. atrox envenomation in the Amazon. Thus, this study aimed to assess the reliability of microhematuria as an inexpensive and simple alternative to detect snake-induced consumption coagulopathy. Methods: We analyzed samples from patients with confirmed B. atrox envenomation in terms of plasma fibrinogen and microhematuria (>3 red blood cells per high power field) in order to access the reliability of microhematuria to detect snakebite-induced coagulopathy, within 12 hours from admission. Results: A total of 186 patients were recruited. From the total, 85.5% of patients had hypofibrinogenemia and only about 50% (n = 94) had a microscopic examination of urine within 12 hours where microhematuria was present in 39 (41.5%). Diagnostic performance showed 38.6% sensitivity and 36.4% specificity (cutpoint 200 mg/dL). No clear association was seen between microhematuria and hypofibrinogenemia (r: -0.10; p: .34). Conclusion: In this study, microhematuria presented poor diagnostic performance to detect coagulopathy. Further studies are necessary to screen inexpensive and simple alternative diagnostic tools.

Tiger snake (Notechis scutatus) envenomation in a horse.

BACKGROUND: A 7-year-old Thoroughbred gelding presented with muscle fasciculation, reluctance to move, profuse sweating, tachycardia, tachypnoea and a localised, unilateral swelling on the muzzle. History and physical examination were suggestive of snake envenomation. METHODS: A sandwich ELISA for the detection of snake venom was performed on serum and urine samples. RESULT: The test performed on urine confirmed a diagnosis of tiger snake envenomation. CONCLUSION: The response to treatment with antivenom and supportive medical therapy was excellent.

Evaluation of snake envenomation-induced renal dysfunction in dogs using early urinary biomarkers of nephrotoxicity.

Renal dysfunction in dogs envenomed by poisonous snakes is currently detected using traditional serum and urinary biomarkers such as creatinine and proteinuria. However, these markers lack sensitivity at the early stages of renal dysfunction and their diagnostic accuracy is affected by pre-analytical factors commonly occurring in these dogs, such as haemolysis and haemoglobinuria. Early detection of renal dysfunction would allow for the identification of dogs requiring intensive treatment and monitoring and may help inform prognosis. The aim of this study was to evaluate the performance of several novel urinary biomarkers of glomerular dysfunction, namely, urinary albumin (uAlb), immunoglobulin G (uIgG) and C-reactive protein (uCRP) and of proximal tubular dysfunction (urinary retinol binding protein (uRBP)) compared to traditional end points in dogs with renal damage caused by snake envenomation. Biomarker results were compared between 19 dogs bitten by snakes producing either neurotoxins or cytotoxins and 10 clinically healthy controls. uAlb, uIgG, and uRBP were significantly increased in snake-envenomed dogs at presentation compared to controls, whereas only uIgG and uCRP were significantly elevated 24h post-envenomation. The urinary protein:creatinine ratio was also increased in envenomed dogs compared to controls, but because of the presence of haematuria and haemoglobinuria, differentiation between pre-renal and renal proteinuria was not possible. The results showed that these novel urinary biomarkers may assist in better detecting renal dysfunction in dogs envenomed by poisonous snakes at the acute disease stage compared to traditional laboratory endpoints.

Development of a double sandwich fluorescent ELISA to detect rattlesnake venom in biological samples from horses with a clinical diagnosis of rattlesnake bite.

Rattlesnake bites in horses are not uncommon and the clinical outcomes are widely variable. Treatment of horses with anti-venom is often cost prohibitive and could have negative consequences; therefore, the development of a quantitative test to determine if anti-venom therapy is indicated would be valuable. The objective of this study was to develop an ELISA to detect rattlesnake venom in biological samples from clinically bitten horses. Nineteen horses were enrolled in the study. Urine was available from 19 horses and bite site samples were available from 9 horses. A double sandwich fluorescent ELISA was developed and venom was detected in 5 of 9 bite site samples and 12 of 19 urine samples. In order to determine if this assay is useful as a guide for treatment, a correlation between venom concentration and clinical outcome needs to be established. For this, first peak venom concentration needs to be determined. More frequent, consistent sample collection will be required to define a venom elimination pattern in horses and determine the ideal sample collection time to best estimate the maximum venom dose. This report describes development of an assay with the ability to detect rattlesnake venom in the urine and at the bite site of horses with a clinical diagnosis of rattlesnake bite.

Proteomic profiling of dog urine after European adder (Vipera berus berus) envenomation by two-dimensional difference gel electrophoresis.

Between April and September every year, many dogs in Finland are bitten by Vipera berus berus, also known as the European adder, the only venomous snake in the area. Exposure to snake bite venom causes local and systemic symptoms and in severe cases can lead to death. Urine samples were collected from four dogs bitten by V. berus berus and treated in the intensive care unit of the Veterinary Teaching Hospital at the University of Helsinki. The inclusion criteria were a strong suspicion of an adder bite no more than two days before admission and clinical signs of an adder bite. Exclusion criteria were defined as ongoing treatment with glucocorticoids or a known history of liver or kidney diseases. Six privately owned, healthy dogs were obtained as controls. Samples were subjected to 2D-DIGE analysis. Image analysis was performed with DeCyder 7.0 2D software, and protein spots demonstrating a minimum 1.5-fold difference in average spot volume ratios between envenomed and control dogs with a Student's t-test p-value of less than 0.05 were picked and identified using LC-MS/MS. In 2D-DIGE analysis, seven proteins were significantly (p < 0.05) over-expressed in the urine of dogs bitten by V. berus berus compared to the control group. From these, five proteins were identified: beta-2-microglobulin (b2MG), alpha-1-antitrypsin (AAT), albumin, fetuin-B and superoxide dismutase (SOD1). Results indicate that envenomation by V. berus berus alter the urinary protein profile in dogs.

An examination of serial urinalyses in patients with North American crotalid envenomation.

STUDY OBJECTIVE: To examine the incidence of abnormal urinalyses after rattlesnake envenomations and its association with bite severity and antivenom administration. METHODS: A retrospective review of data collected in a prospective manner for an experimental crotalid antivenom trial. Subjects were individuals with minimal to moderate North American crotalid envenomations. Incidence and characterization of abnormal urinalysis after crotalid envenomation is presented. Additionally, the relationship of abnormal urinalysis to bite severity is examined. A preliminary test of antivenom protein urinalysis interference was also conducted. RESULTS: Forty-three percent of the urinalyses reported prior to antivenom treatment had abnormalities. Thirty-three of 41 subjects (80%) had an abnormal urinalysis, defined as the presence of cells, blood, glucose, or protein, at some time during the 2-week period following envenomation. All but 3 of these subjects had urinalyses which returned to normal by 2 weeks postenvenomation. Fifteen of 22 subjects (68%) with minimal envenomations had an abnormal urinalysis at some time following envenomation, while 18 of 19 subjects (95%) with moderate envenomations had abnormal urinalyses (p < 0.05). In addition, high concentrations of antivenom added to urine were found to produce a positive urine dipstick test for protein. CONCLUSION: In our study of patients with minimal to moderate North American crotalid envenomations, there was a high incidence of abnormal urinalyses. The urine abnormalities tended to be more common with increased bite severity and more frequent during the first few hours following envenomation. Antivenom appearance in the urine could be responsible for some of our findings.

Levels of therapeutic antivenin and venom in a human snakebite victim.

An enzyme-linked immunosorbent assay was used to measure the levels of therapeutic antivenin (Antivenin [Crotalidae] Polyvalent, Wyeth-Ayerst) in serum and the levels of venom in the urine of a patient bitten by a western diamondback rattlesnake (Crotalus atrox). Serum and urine samples were taken on admission, during hospitalization, and during follow-up until 5 months after the bite. Photographs were taken of the bite site on admission to the hospital and during follow-up. Serum levels of therapeutic antivenin were highest between 1 and 3 days after the bite but were still detectable 46 days as well as 4 months after the bite. Crotalus atrox venom was detectable in the urine 4 days after the bite and was still measurable 6 days after the bite.

Urinary NAG as an early indicator of renal damage in Russell's viper bite envenomation.

Urinary N-acetyl-beta-D-glucosaminidase (NAG; beta-N-acetylglucosaminidase; EC 3.2.1.30), blood urea, serum creatinine and creatinine clearance were measured in 23 patients bitten by Russell's viper (Daboia russelii siamensis), with systemic envenomation, at different time intervals after the bite during clinical observation for 5 d. Activities of urinary NAG were compared with these indicators of renal function in 3 clinical conditions: non-oliguric acute renal failure (ARF), oliguric ARF not requiring peritoneal dialysis, and oliguric ARF requiring peritoneal dialysis. Cut-off values to identify the 3 conditions were established. When the times of onset of the conditions, indicated by the cut-off values, were compared, urinary NAG was generally found to be the earliest indicator of renal damage. In each type of ARF, the urinary NAG level was abnormal before changes in the values of the other indicators of renal function. It may be possible to predict the types of ARF within 2 h after the bite by measurement of urinary NAG.

Some toxicity thresholds for the clinical effects of common tiger snake (Notechis scutatus) envenomation in the dog.

Common tiger snake (Notechis scutatus) venom was administered experimentally to dogs at doses from 0.25 lethal dose (LD) to 20 LD. Haemolysis and increased creatine kinase values occurred rapidly after injection of sublethal (subparalytic) doses, but the clotting time of blood was extended and blood became incoagulable only when dogs were dosed with 10 LD or more of venom. Haemolysis, although of a low threshold of toxicity, was not severe and should not greatly affect the lethality of the venom. Coagulopathy is a sign that the dog has been lethally envenomed and will need to be given antivenom if skeletal muscle paralysis is to be overcome.

Urinary fibrin (-ogen) degradation products in Russell's viper (Daboia russelii siamensis) bite victims.

Serum and urine concentrations of fibrin (-ogen) degradation products (FDP) were estimated in 20 proven Russell's viper bite (RVB) cases with severe defribination. All patients had similar degrees of high serum FDP levels. However, the ten who developed into acute renal failure (ARF) had significantly (p < 0.001) higher urinary FDP levels than those who did not. The urinary FDP levels of ARF cases increased correspondingly with high serum FDP levels but not in cases without ARF. Serial comparison of serum and urinary FDP levels in RVB cases with severe defibrination may be of value in predicting the likelihood of developing ARF. The present study favored disseminated intravascular coagulation as the main cause of ARF in Myanmar RVB cases.

Envenoming by viper bites in France: clinical gradation and biological quantification by ELISA.

Viper bites are frequent in France but the evaluation of the severity of envenomings and consequently patient treatment has not yet been properly evaluated. The purpose of this study was to measure venom antigens in blood and/or urine of bitten patients and to establish a quantitative relationship with clinical observations. A prospective enquiry was conducted in 1990 in France to collect epidemiological, clinical and biological data from hospitals. Urine and blood samples were tested for their content of Vipera aspis venom antigens by a sandwich enzyme linked immunosorbent assay (ELISA). One hundred and two charts were analysed, from patients presenting documented viper bites. Oedema was the prominent local feature (81 cases). Systemic signs consisted of vomiting and/or diarrhoea (22 cases), slight or severe hypotension (15 cases), shock (2 cases) and bleeding (1 case). A relationship was observed between these systemic signs and the extent of the oedema, which permitted the establishment of a grading scale. Grade 0 (no envenoming) was identified by fang marks and absence of oedema and local reaction; grade 1 (mild envenoming) by local oedema and absence of systemic symptoms; grade 2 (moderate envenoming) by regional oedema and moderate systemic symptoms; and grade 3 (severe envenoming) by extensive oedema and severe systemic symptoms. Quantification of venom antigens in blood or urine of patients by ELISA revealed a significant correlation between clinical signs of envenoming and the level of venom antigens in blood or urine. This indicated that the ELISA test is a useful and predictive tool for clinically grading viper envenomings.

Evidence that Crotalus durissus terrificus (South American rattlesnake) envenomation in humans causes myolysis rather than hemolysis.

The venom of the South American rattlesnake Crotalus durissus terrificus was originally reported to have a pathophysiological activity mainly involving hemolysis and neurotoxicity. The systemic myotoxic action of this venom was demonstrated in 1985. In the present paper we report clinical and laboratory data concerning three patients bitten by C. durissus terrificus and treated at the University Hospital of Ribeirão Preto, University of São Paulo. The normal haptoglobin levels detected in the serum of these patients during the first 48 hr after the accident, as well as the absence of hemoglobin in darkened urine samples as evaluated by immunodiffusion against anti-hemoglobin serum, rule out the occurrence of intravascular hemolysis. These data permit us to conclude that the signs and symptoms observed in human envenomation with C. durissus terrificus are due to a myotoxic and neurotoxic action of the venom.

Russell's viper venom levels in serum of snake bite victims in Burma.

Serum levels of venom antigen were measured using enzyme-linked immunosorbent assay (ELISA) in 38 Russel's viper bite victims before and after administration of 40 ml of monovalent liquid antivenom. Initial serum levels ranged from one with less than 10.0 ng to 290 ng/ml and in one case a level of 75 ng/ml was detected 27 hours after the bite. Serum venom levels after liquid monospecific antivenom therapy indicated that venom clearance was similar in each case to the natural clearance of venom in the absence of antivenom therapy. In one case a venom level of 11.5 ng/ml was detected 66 hours after liquid antivenom therapy whereas in two fatal cases, serum venom levels of 95 ng/ml and 185 ng/ml were detected after the same interval. Failure of complete neutralization of venom is probably the result of loss of potency of antivenom during improper storage. The amount of venom excreted in the urine was not related to initial serum levels.