Red-bellied black snake (Pseudechis porphyriacus ...

Toxicon 117 (2016) 69e75

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Case report

Red-bellied black snake (Pseudechis porphyriacus) envenomation in the dog: Diagnosis and treatment of nine cases

Andrew M. Padula*, Kenneth D. Winkel

Australian Venom Research Unit, Department of Pharmacology and Therapeutics, Faculty of Medicine, Dentistry and Health Sciences, University of Melbourne, 3010, Australia

article info

Article history: Received 29 January 2016 Received in revised form 16 March 2016 Accepted 31 March 2016 Available online 1 April 2016

Keywords: Snake venom Snake antivenom Snakebite Black snake Pseudechis porphyriacus Immunoassays

abstract

The clinical signs, biochemical changes and serum and urine venom concentrations for a series of nine cases of Red bellied black snake [RBBS] (Pseudechis porphyriacus) envenomation in eight dogs seen in a regional Australian veterinary hospital are described. Although the resulting envenomation syndrome was, in most cases, relatively mild and responded rapidly to intravenous administration of a novel bivalent caprylic acid purified whole IgG equine antivenom for tiger (Notechis scutatus) and brown snake (Pseudonaja textilis), one fatality prior to antivenom treatment was recorded. The latter case occurred within 1 h of envenomation prior to receiving antivenom treatment. Intravascular haemolysis, pigmenturia, bite site swelling, lethargy, and generally mild coagulopathy were present in most cases. Detectable RBBS venom specific components were found in serum, bite site swab or urine using a standard sandwich ELISA approach. Serum levels fell within the range previously reported for human RBBS envenomation cases (6e79 ng/ml) whilst bite site and urine samples varied more markedly (8.2 to >5000 ng/ml and 2.2e1300 ng/ml respectively). No venom was detected from serum after antivenom treatment. The envenomation syndrome in dogs is similar to what is described for humans, with the exception of the presence of potentially severe venom induced consumption coagulopathy in one case (aPTT > 300 s and fibrinogen < 0.43 g/L) and potential for fatal outcomes. This series represents the largest and most detailed examination of RBBS envenomation in animals yet reported. It reinforces the emerging view that the potential severity of this envenomation has been underappreciated by veterinary practitioners and highlights the possibility of severe venom induced consumption coagulopathy in canine cases.

? 2016 Elsevier Ltd. All rights reserved.

1. Introduction

The Australian Red-bellied Black snake (RBBS) (Pseudechis porphyriacus) is a large, distinctively coloured and moderately venomous snake distributed widely throughout south-eastern Australia. The RBBS is diurnal and typically found near waterways, swamps and lagoons where it feeds predominantly on frogs (Cogger, 2000). It's venom was first investigated by Charles Martin in the 1890's as a prototype for Australian elapids (Hawgood, 1997) and then, more comprehensively, by Charles Kellaway in the 1930's (Kellaway, 1930). The venom is notably less toxic than that of other Australian elapids with an LD50 of 2.52 mg/kg in 18e21 g mice (Broad et al., 1979) and contains neurotoxins, procoagulants and

* Corresponding author. 26 Howitt Ave, Bairnsdale, Victoria, 3875, Australia. E-mail address: andrew.padula@unimelb.edu.au (A.M. Padula).

0041-0101/? 2016 Elsevier Ltd. All rights reserved.

myotoxins (Pearn et al., 2000). The venom of the RBBS is effectively neutralised by antivenom directed against tiger snake (Notechis scutatus) venom (Best and Sutherland, 1991).

Despite its wide distribution there are very few published reports of envenomation in animals (Gordon, 1958; Heller et al., 2006). Surveys of veterinarians in Australia have revealed that RBBS envenomation is relatively common in certain geographical areas. For example, in one study 44.6% of animal snakebite cases treated in 253 veterinary clinics in New South Wales were for presumed RBBS envenomation (Heller et al., 2005). The most common clinical findings in such presumed, but not formally confirmed cases, were pigmenturina, weakness, ataxia and salivation (Heller et al., 2005). To address the paucity of veterinary clinical data on this envenomation type, and to complement recent human findings, we examined RBBS envenomation in dogs treated at a regional Victorian veterinary hospital. Hence this report describes the clinical features, serum biochemistry, haematological,

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A.M. Padula, K.D. Winkel / Toxicon 117 (2016) 69e75

coagulation status, venom concentrations and treatment outcomes, in nine cases of RBBS envenomation in eight dogs.

2. Materials and methods

2.1. RBBS venom specific ELISA

The concentration of RBBS venom was measured in clinical samples (serum, urine, bite site swab) using a sandwich ELISA. The ELISA was similar to that previously utilised for measuring taipan venom (Churchman et al., 2010; Kulawickrama et al., 2010). RBBS venom specific antibodies were purchased from a commercial supplier (Harry Perkins Institute, Perth, Western Australia). The rabbit anti-RBBS IgG was purified by passing the crude rabbit serum through a Protein G column and eluting the bound IgG. A biotin label was applied to the rabbit anti-RBBS IgG preparation using a commercial kit (EZ-Link, Pierce, USA). The RBBS ELISA was performed by coating 96-well polystyrene high binding micro-

plates (MaxisorpTM, Nunc, USA) with 100 mL/well of rabbit antiRBBS IgG at 10 mg/mL in carbonate coating buffer pH 9.6. Plates

were incubated at room temperature for 3 h and then placed in a refrigerator overnight at 4. Next day the plates were washed

three times with PBS-T20 and unbound sites blocked with 300 mL/

well of ELISA blocking buffer consisting of PBS-T20 ? 0.5% BSA (Bovostar, Bovogen, Australia). Controls, standards and test sam-

ples were pipetted into each well in a volume of 100 mL and

incubated for 30 min on a plate shaker at 600 rpm for 30 min. Unbound venom was removed by washing plates three times as

described above. Next, 100 mL of the secondary biotin labelled rabbit anti-RBBS (0.15 mg/mL) in blocking buffer was pipetted into

each well and incubated as described above. Following incubation, unbound biotin antibody was then removed by washing the plate three times. Streptavidin-HRP (Thermo Fisher, Australia) was then added at a 1:40,000 dilution in blocking buffer and incubated the same as for the secondary antibody. Plates were washed for a final

time and 50 mL of TMB (Ultra TMB, Thermo Fisher, Australia) was

added to each well and colour allowed to develop for 4e10 min.

The enzyme reaction was then stopped by addition of 50 mL of 10%

sulphuric acid. Plates were read within 10 min in a Tecan Sunrise microplate reader at 450 nm. Unknown samples were interpolated against the standard curve using a 5-point fitted equation computed from commercial software (MagellanTM 7.2, Tecan, Austria).

Serum, urine and bite site swabs were initially diluted to 10% in ELISA blocking buffer and applied to Row 1 on the microplate. Doubling dilutions were then prepared down the plate to provide a range of sample concentrations from 1:10 to 1:1280. A standard curve was run in duplicate on each plate consisting of RBBS venom (Venom Supplies Pty Ltd, Tanunda, South Australia) dissolved in ELISA blocking buffer to a final concentration of 5 ng/mL.

Negative control samples were run in each assay run consisting of pooled normal dog urine and serum collected from ten nonenvenomed dogs. Raw optical density (OD) values of unknown samples that were below the OD of the negative control for each matrix were not used for calculations. The mean OD of eight blank wells was subtracted from the OD reading of each standard, control or test sample well. The sensitivity of the RBBS venom ELISA was 0.30 ng/mL. The RBBS ELISA was evaluated for cross reactivity with Tiger snake (Notechis scutatus) and Eastern brown snake (Pseudonaja textilis) venoms by assaying doubling dilutions of these venoms starting at 1000 ng/mL. The RBBS venom ELISA was found to be highly specific for RBBS venom with only 0.7% cross-reactivity with Tiger and 5000 ng/mL). Serum RBBS venom concentration at presentation was 12 ng/mL, urine 1090 ng/mL and venom was undetectable in the serum sample collected at 15-min post-antivenom.

14 h after antivenom administration the dog appeared normal and was sent home. At 36 h post-presentation the dog returned because it was again lethargic. A full clinical haematology and biochemistry profile was performed that revealed the PCV had reduced to 19% (37e55) with haemoglobin 54 g/L (115e180) and evidence of regenerative anaemia with 6.9% reticulocytes. Examination of the blood smear revealed morphological abnormalities of the red blood cells including rouleaux formation 3?, polychromasia? and a mild thrombocyotopaenia. Serum biochemistry revealed CK 1363 U (300 s) demonstrating that even larger animals should be considered vulnerable to this snake's venom. The comparison here, of urine with other tissues, also shows that the latter is likely to have at least as high a venom concentration as that of serum. Hence, urine can be confidently sampled for immunoassays to aid the diagnosis and management of RBBS bite in dogs.

The venom of the RBBS is dominated by the presence of large quantities of the phospholipase A2 isoenzyme pseudexin that has neurotoxic effects on mouse diaphragm tissues (Vaughan et al., 1981) and chick biventer cervis nerve-muscle preparations (Ramasamy et al., 2004). Given the rapid clinical deterioration, it seems possible that the high levels of these neurotoxins, combined with small body weight, contributed to the rapid death of the dog in case 8. Alternatively, early cardiovascular collapse, strongly associated with the presence of prothrombin activators in other

A.M. Padula, K.D. Winkel / Toxicon 117 (2016) 69e75

75

Australian snake venoms (Allen et al., 2012), may be responsible for the rapid fatality. This is particularly plausible as no dog showed evidence of even mild neurotoxicity whilst the animal most affected by coagulopathy (case 5), also developed early post-bite `collapse'. Indeed, as case 5 and the earlier fatal case (Heller et al., 2006) demonstrate, both coagulopathy and myohaemoglobinurictype acute renal failure are potentially lethal aspects of this envenomation in dogs.

The major limitation of this study was the small sample size resulting in significant variation in animal size (6e38 kg), time to presentation (30 min to at least 12 h post-bite), venom levels (urine levels from 2.2 to 1300 ng/ml). Despite this degree of variation, a normal aspect of veterinary practice, there was a remarkable consistency to the observed envenomation syndrome. Clearly it would be advantageous to enrol a larger series of animals and collect a larger number of bite site, serum and urine samples to more formally assess the relationship between venom antigenemia across the various tissue compartments. A second limitation is the question as to how the tested antivenom compares to older tiger/ brown bivalent antivenoms such as the CSL Limited (now Sequirus Limited) or Summerland Serums that are commonly used in clinical practice (Ong et al., 2015). The logical next step is a non-inferiority trial directed against various clinical end points for all three products. Given the apparent importance of RBBS envenomation in Australian veterinary practice (Heller et al., 2005), this seems a feasible and worthwhile goal.

In conclusion, although the envenomation syndrome in dogs caused by RBBS envenomation is usually mild in respect of clinical signs and coagulation disturbances, and is highly responsive to antivenom therapy, severe manifestations and complications can occur. Veterinary practitioners should be vigilant for such possibilities even after the administration of appropriate doses of antivenom.

Acknowledgements

Thank you to staff at Bairnsdale Animal Hospital for assisting with treatment of snakebite cases. Funding from the National Health and Medical Research Council (Australia) (TP701736) supported the contribution of one author (KDW) to this publication.

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