MURDOCH RESEARCH REPOSITORY.

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1 MURDOCH RESEARCH REPOSITORY This is the author's final version of the work, as accepted for publication following peer review but without the publisher's layout or pagination. Peacock, R.E., Hosgood, G., Swindells, K.L. and Smart, L. (2013) Aplysia giganteatoxicosis in 72 dogs in Western Australia. Australian Veterinary Journal, 91 (7). pp Copyright 2013 The Authors. Australian Veterinary Journal It is posted here for your personal use. No further distribution is permitted.

2 Aplysia gigantea toxicosis in 72 dogs RE Peacock*, G Hosgood, KL Swindells and L Smart * Corresponding author School of Veterinary and Biomedical Sciences, Murdoch University, 90 South Street, Murdoch, Western Australia, 6150; r.peacock@murdoch.edu.au Objectives This study aims to: 1) confirm a temporal association between exposure to the sea hare Aplysia gigantea and the development of a neurotoxicosis in dogs, 2) further characterise the clinical signs in dogs with this suspected toxicosis, and 3) report the treatment and outcome of dogs with this suspected toxicosis. Method The medical records from four veterinary practices within the Geraldton region of Australia were searched for dogs that had been exposed to Aplysia gigantea and were subsequently presented to a veterinarian during the period of January 2001 to March Signalment, exposure history, clinical signs, treatment and outcome were recorded. Results Seventy-two dogs met the inclusion criteria. Clinical signs included ptyalism, emesis, ataxia, hyperaesthesia, tremors, muscle fasciculations, seizures, nystagmus and respiratory distress. Thirty dogs did not have abnormal clinical signs at presentation. Sixty-nine dogs presented during January to April. Treatment included gastrointestinal and dermal decontamination, and supportive management of seizures, tremors and muscle fasciculations. Sixty-five dogs survived to discharge, four dogs died, and three were euthanised. Information from subsequent examinations was available for 57 dogs and no long-term complications were reported. Conclusions Exposure to Aplysia gigantea was temporally associated with the development of neuroexcitatory clinical signs in dogs. Gastrointestinal and respiratory signs also occurred in some dogs. Dogs with suspected toxicosis mostly presented in the months from January to April. The proportion of dogs that died or were euthanised due to worsening clinical signs was 10%. Keywords: Aplysia, seizure, beach, sea hare, toxicosis, dog. Introduction Veterinarians practicing along the Western Australian coastline near Geraldton have observed that dogs who have had contact with the sea hare Aplysia gigantea can develop neuroexcitatory signs and some dogs die. 1 The normal life cycle of the sea hare results in hundreds of dead A. gigantea washing up on Geraldton beaches periodically. 2 These events coincide with anecdotal reports of dogs developing neuroexcitatory clinical signs after being at local beaches. 1 Sea hares are herbivorous marine molluscs and are a type of sea slug. A distinguishing feature of sea hares is the presence of a pair of large parapodial lobes, which Aplysia species can use for swimming. 3 Aplysia gigantea is black or dark brown in colour and is one of the largest sea hares, growing up to 60 cm long and weighing up to 2 kg. 3 The documented range of A. gigantea includes the coastal waters of the southwestern corner of Western Australia between Duke of Orleans Bay, east of Esperance, to Bluff Point, north of Geraldton. 2 Anecdotal reports of suspected A. gigantea toxicosis in dogs are available. 1,4 The purpose of this study was to: 1) confirm a temporal association between exposure to the sea hare A. gigantea and the development of neuroexcitatory clinical signs in dogs, 2) further characterise the clinical signs in

3 dogs with suspected toxicosis after exposure to A. gigantea, and 3) report the treatment and outcome of dogs with suspected toxicosis after exposure to A. gigantea. Materials and Methods The medical records from four veterinary practices a,b,c,d within the Geraldton region were searched using the keywords: tremor, seizure, shaking, fit, convulsion, hyperaesthesia, toxin, poison, nudibranch, marine, beach, blobbie, Aplysia, Spanish (dancer), snail, slug and hare. Records dated between January 2001 and March 2011 were searched for cases matching the inclusion criteria. Cases were included if a dog had been observed by the owner to have direct contact with A. gigantea, either through licking or rolling, or to have ingested A. gigantea. Information retrieved from records included signalment, body weight, exposure history including ingestion or contact with A. gigantea and time from exposure to the onset of clinical signs, month of the year, owner observations prior to presentation, physical examination findings, treatment, length of hospitalisation, outcome including survival to discharge, death or euthanasia, and details of subsequent examinations. Numerical data were summarised as a median and range. Categorical data were summarised as a frequency. Physical examination findings were divided according to neurologic, gastrointestinal and cardiopulmonary system involvement where possible. A commercially available statistical software package e was used to summarise data and perform calculations. Results A search of the keywords returned 6,571 records of which 72 cases met the inclusion criteria. The median age of the dogs was 2.5 years (n=64, range 2 months to 13 years). The median weight was 23.4 kg (n=63, range kg). Thirty-four female dogs (22 neutered) and 34 male dogs (21 neutered) were recorded. Breed was recorded for 67 dogs and included mixed-breed dogs (n=25), Labrador retriever (17), Australian cattle dog (4), Staffordshire bull terrier (4), and golden retriever (3). Eleven other breeds were represented by one or two dogs each. Forty dogs had abnormal clinical signs that were observed by the owners prior to presentation. Forty two dogs had abnormal findings on physical examination at presentation. One dog that had an abnormality observed by the owner was normal on physical examination at presentation. Three dogs were reported by the owners to have been normal prior to presentation but had abnormal findings on physical examination at presentation. The number of dogs having contact with an A. gigantea was 37, and the number of dogs that had ingested an A. gigantea was 35. The owners reported tremors (n=17), shaking (12), vomiting (11), seizures (8), ataxia (7), drooling (5), diarrhoea (2), disorientation (1), and oral discomfort (1) in the dogs prior to presentation to the veterinarian. The median time from direct contact to the onset of the signs was 1.75 hours (n=14, range hours). The median time from ingestion to the onset of the signs was two hours (n=26, range hours). Dogs were presented in January (n=13), February (29), March (18), April (9), May (1), June (1), and July (1). Clinical signs recorded by veterinarians included tremors (n=29), ataxia (11), hyperaesthesia (10), muscle fasciculation (6), panting or tachypnoea (6), vomiting (5), ptyalism (5), cardiopulmonary arrest (4), seizures (3), and nystagmus (3) (Tables 1, 2, and 3). Pigmenturia was present in two dogs. The median rectal temperature was 40.0 o C (n=18, range o C).

4 Gastrointestinal decontamination was performed in 15 dogs including emesis (n=12), gastric lavage (2) and activated charcoal administration (2). Material resembling A. gigantea, such as dark slimy material, was present in the vomitus of four dogs and the gastric lavage fluid of one dog. Only one dog received both gastric lavage and activated charcoal. Dermal decontamination via bathing in a mild detergent was performed on 10 dogs at presentation. All dogs displaying signs of toxicosis received more than one drug (Table 4). The most frequent drugs used were intravenous fluids (n=28), diazepam (22) and pentobarbitone (13). The number of dogs that were discharged immediately after consultation was 22 (Table 5). None of these dogs were reported to have returned for repeat consultation. Duration of hospitalisation was less than 24 hours for 64 dogs (Table 5). The number of dogs that survived to discharge was 65 and all were recorded as clinically normal at discharge. Of the remaining seven dogs, two were dead on arrival, two died within 12 hours of presentation due to worsening of clinical signs despite treatment, and three were euthanised within 24 hours due to worsening of clinical signs despite treatment. Three dogs that died had received cardiopulmonary and cerebral resuscitation. All dogs that died had been seen to ingest A. gigantea. Information from subsequent examinations was available for 57 dogs. No owners reported evidence of continued illness that could be related to suspected A. gigantea toxicosis. One owner reported a small amount of skin sloughing in one dog, which the attending veterinarian had suspected was due to extravasation of pentobarbitone. Discussion This study confirms that neuroexcitatory signs in dogs are temporally associated with exposure to A. gigantea. The neuroexcitatory signs are consistent with those seen in other domestic species that have had toxicosis induced by chemical extracts from related sea hares. 5,6,7,8 Toxicosis may be due to the presence of cholinomimetic chemicals in the digestive gland and such chemicals have been isolated from other Aplysia species. 9,10,11,12,13 Gastrointestinal and respiratory abnormalities were also identified in the present study. It is possible that dogs with other clinical signs may have been missed as a number of the keywords searched were based on anecdotally reported clinical signs. 1 Attempts were made to minimise selection bias by using a number of keywords that were not clinical signs. This selection bias may also have led to an over-representation of clinical signs that were also keywords. An under-representation of some clinical signs may also have occurred due to inadvertent omission from the medical records. Tremors were the most frequent neurological manifestation of toxicosis in this study. Diazepam and pentobarbitone were among the most frequently used drugs despite the low frequency of seizures. Muscle tremors due to some neuroexcitatory poisons have been reported to be poorly responsive to benzodiazepines and barbiturates. 14,15,16 Muscle relaxants such as guaifenesin and methocarbamol may be more effective at controlling tremors in these circumstances. 14,15 Abnormal respiratory signs were reported in some dogs. Several studies have reported respiratory distress prior to death in mice injected intraperitoneally with extracts from other Aplysia species. 7,8 The cause of respiratory distress is unknown, however, bronchoconstriction may play a role if the toxin has cholinomimetic effects. 9,10,11,12,13 In select cases, the use of a bronchodilator may be justified. Theophylline has been anecdotally used in dogs with suspected A. gigantea toxicosis and respiratory distress, 1 with a view that it improved the respiratory signs. Theophylline was used in four dogs in this study, only one of which had a recorded respiratory abnormality. A β-adrenergic receptor agonist, such as terbutaline, may be a better choice compared to theophylline, as it should reverse bronchoconstriction regardless of the cause. Atropine was used in three dogs in this study,

5 although the indication for the administration was not recorded. Atropine administration may be beneficial in select cases where ptyalism is present, or respiratory distress due to bronchoconstriction and bronchorrhoea is suspected, however atropine should not be expected to reverse the central or peripheral nervous system signs.two studies on the use of atropine in isolated muscle affected by toxins from other Aplysia species show conflicting results. 10,11 The proportion of dogs with clinical signs that died or were euthanised approached 10%. The cause of cardiopulmonary arrest was not known for any dog that died naturally. The cause of death may be a direct result of the toxin, or may be due to secondary complications such as hyperthermia, hypoglycaemia, or hypoxaemia. No prospective toxicological studies have been performed to isolate toxic compounds from A. gigantea. Studies investigating the toxic effects of chemical extracts from other sea hare species only report clinical signs of the toxicosis and the cause of death is unknown. 5,6,7,8 The reason stated for euthanasia, where performed, was a worsening of clinical signs despite treatment. This study may misrepresent the number of dogs that died, or the proportion of dogs that developed clinical signs, after exposure to A. gigantea as there may be a population of dogs with exposure that never presented to a veterinarian. In this study, there was variability in the severity of clinical signs among dogs. Some dogs had only mucous membrane or dermal contact with A. gigantea and developed severe clinical signs, whereas other dogs ingested A. gigantea and developed no clinical signs. This could indicate either a variance in host susceptibility, a variable state of toxicity among an A. gigantea population, or variable levels of toxicity between different areas of the sea hare itself. One research group studying chemicals from A. kurodai found that the type and concentration of chemicals varied depending on when and where the animals were collected, 17 supporting the theory that a variable state of toxicity may exist. The same variability in toxicity may also explain the range in time from contact with, or ingestion of, A. gigantea to the onset of clinical signs reported in this study. Most dogs in this study presented in the months from January to April. This may be due to a seasonal increase in the toxicity of A. gigantea, but is also likely associated with A. gigantea mass mortality events, which occur during this time of year. Despite marine mass mortality events becoming increasingly common in a number of coastal environments, very little is documented about the underlying cause. 18 Mass mortality events appear to be part of the normal life cycle of A. gigantea and field studies have shown that other Aplysia species rarely live beyond a year. 19 There are a number of theories as to why mass mortality events may vary from year to year, with most linked to environmental factors such as rough weather, increases in population temporally associated with warmer water temperature or algal blooms, and unusually hot weather combined with low tides or regions with broad sand flats. 19,20,21,22,23 Predicted global environmental change has been implicated in coastal environments with increased marine mass mortality events. 24 In this study, confirmation of exposure to an A. gigantea relied upon accurate identification of this sea hare by the dog owner. Aplysia gigantea are large and distinct marine animals and a common finding on Geraldton beaches, so the likelihood of a Geraldton resident correctly identifying these animals is high. A visitor to Geraldton may not accurately identify an A. gigantea specimen, particularly when there has been rough weather leading to seaweed and other marine life washing up en masse on local beaches. Therefore, a proportion of dogs in this study may not have had exposure to A. gigantea if this sea hare was incorrectly identified. Similarly, this study does not include dogs where exposure to A. gigantea occurred but was incorrectly identified and recorded in

6 the computer records. These dogs would have either been missed by the keyword search or excluded due to lack of confirmed A. gigantea exposure being recorded. Treatment was given at the attending veterinarian s discretion and most commonly included supportive care and drugs aimed at controlling neuroexcitatory signs. A high proportion of dogs with clinical signs received intravenous fluids. Intravenous fluid therapy may assist with the renal excretion of the toxin but should be titrated according to individual needs. Due to the proportion of dogs with respiratory signs in this study, close monitoring for development of pulmonary oedema is recommended for dogs with this suspected toxicosis that are receiving intravenous fluids. Blood pressure should be monitored closely as experiments using Aplysia toxins have shown hypertensive and hypotensive effects that may influence treatment plans. 10,25 Few dogs had toxin decontamination, either dermal or gastrointestinal, in this study. Most of the gastric contents removed via induction of emesis or gastric lavage, did not contain material resembling A. gigantea. Anecdotal evidence suggests that emesis, when possible, may be a more effective gastric decontamination procedure than lavage, as pieces of A. gigantea, when present, may be too large to come through the stomach tube. 1 This study would suggest that dogs with exposure to A. gigantea are at high risk of developing life-threatening toxicosis, and therefore gastrointestinal decontamination in dogs that have ingested A. gigantea would be justified. Similarly, it would be justified bathing dogs with dermal exposure. Exposure to Aplysia gigantea at beaches in the Geraldton region was temporally associated with the development of neuroexcitatory clinical signs in dogs. Gastrointestinal and respiratory clinical signs also occurred in some dogs. Dogs with suspected toxicosis most commonly present in the months from January to April. The proportion of dogs that died or were euthanised due to worsening clinical signs was 10%. Acknowledgments The authors acknowledge the access to medical records provided by the Chapman Animal Hospital, Dongara Veterinary Hospital, Midwest Veterinary Centre and Sanford Veterinary Clinic, and the assistance of Mr Clayton Bryce from the Western Australian Museum. a Chapman Animal Hospital. b Dongara Veterinary Hospital. c Midwest Veterinary Centre. d Sanford Veterinary Clinic. e SAS v9.3, SAS Institute, Cary, NC, USA. References 1. Ingham G. Nudibranch poisoning - cases in dogs. Post Graduate Foundation of The University of Sydney (now the Centre for Veterinary Education). Control & Therapy Series 2002;C&T No. 4312, Mailing 224: Wells FE. A redescription of the sea hare Aplysia gigantea Sowerby, Journal of the Malacological Society of Australia 1986;7: Wells FE, Bryce CW. Sea slugs of Western Australia. Western Australian Museum, Perth, 1993: Sea Slug Forum. Dog-poisoning Sea Hares make the news Accessed 6 th August 2012.

7 5. Flury F. Über das Aplysiengift. Arch Exp Pathol Pharmakol 1915;79: Watson M. Midgut gland toxins of Hawaiian sea hares - I. Isolation and preliminary toxicological observations. Toxicon 1973;11: Winkler LR. Preliminary tests of the toxin extracted from Californian sea hares of the genus Aplysia. Pac Sci 1961;15: Melo VM, Fonseca AM, Vasconcelos IM, Carvalho AF. Toxic, antimicrobial and hemagglutinating activities of the purple fluid of the sea hare Aplysia dactylomela Rang, Braz J Med Biol Res 1998;31: Blankenship JE, Langlais PJ, Kittredge JS. Identification of a cholinomimetic compound in the digestive gland of Aplysia californica. Comp Biochem Physiol 1975;51C: de Freitas JC. Action of crude and fractioned homogenates of the midgut gland of the sea hare Aplysia brasiliana rang, 1828 on some cholinoceptive structures. Comp Biochem Physiol 1977;56C: Kerchove CM, Markus RP, de Freitas JC, Costa-Lotufo LV. Evaluation of the cholinomimetic actions of trimethylsulfonium, a compound present in the midgut gland of the sea hare Aplysia brasiliana (Gastropoda, Opisthobranchia). Braz J Med Biol Res 2002;35: Winkler LR, Tilton BE, Hardinge MG. A cholinergic agent extracted from sea hares. Arch Int Pharmacodyn Ther 1962;137: Winkler LR, Tilton BE, Hardinge MG. Predation on the Californian sea hares, Aplysia californica, Cooper, by the solitary grate anemone, Anthopleura xanthogrammica, Brandt, and the effect of the sea hare toxins and acetylcholine on anemone muscles. Pac Sci 1962;16: Hiromori T, Nakanishi T, Kawaguchi S, et al. Therapeutic effects of methocarbamol on acute intoxication by pyrethroids in rats. J Pesticide Sci 1986;11: Lowes NR, Smith RA, Beck BE. Roquefortine in the stomach contents of dogs suspected of strychnine poisoning in Alberta. Can Vet J 1992;33: Boysen SR, Rozanski EA, Chan DL, et al. Tremorgenic mycotoxicosis in four dogs from a single household. J Am Vet Med Assoc 2002;221: Yamamura S, Hirata Y. Structures of aplysin and aplysinol, naturally occurring bromo-compunds. Tetrahedron 1963;19: Cebrian E, Uriz MJ, Garrabou J, Ballesteros E. Sponge mass mortalities in a warming mediterranean sea: are cyanobacteria-harboring species worse off. PLoS One 2011;6:e Audesirk TE. A field study of growth and reproduction in Aplysia californica. Biol Bull 1979;157: Plaut I, Borut A, Spira ME. Seasonal cycle and population dynamics of the sea hare Aplysia oculifera in the northern Gulf of Eilat (Aqaba), Red Sea. J Moll Stud 1998;64: Crisci C, Bensoussan N, Romano J-C, Garrabou J. Temperature anomalies and mortality events in marine communities: insights on factors behind differential mortality impacts in the NW Mediterranean. PLoS ONE 2011:6:e23814-e Fukui Y, Saitoh S, Sawabe T. Environmental determinants correlated to Vibrio harveyi-mediated death of marine gastropods. Environ Microbiol 2010;12: Russell J, Phillips N. Species-specific vulnerability of benthic marine embryos of congeneric snails (Haminoea spp.) to ultraviolet radiation and other intertidal stressors. Biol Bull 2009;217: Coma R, Ribes M, Serrano E, et al. Global warming-enhanced stratification and mass mortality events in the Mediterranean. PNAS 2009;106:

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9 Table 1. Frequency of neurologic signs in 72 dogs exposed to Aplysia gigantea Clinical sign Number of dogs Tremors 29 Ataxia 11 Hyperaesthesia 10 Muscle fasciculation 6 Seizures 3 Agitation 3 Nystagmus 3 Mydriasis 2 Delayed pupillary light reflexes 1 Temporary blindness 1 None 36 Table 2. Frequency of gastrointestinal signs in 72 exposed to Aplysia gigantea Clinical sign Number of dogs Vomiting 5 Ptyalism 5 Diarrhoea 2 None 61 Table 3. Frequency of cardiopulmonary signs in 72 dogs exposed to Aplysia gigantea Clinical sign Number of dogs Tachypnoea 6 Cardiopulmonary arrest 4 Pulmonary crackles 2 Dyspnoea 2 None 61 Table 4. Frequency of treatments used for 72 dogs exposed to Aplysia gigantea Treatment Number of dogs Intravenous fluids 28 Diazepam 22 Pentobarbitone 13 Corticosteroids 8 Antibiotics 7 Acepromazine 5 Frusemide 5 Active cooling 4 Butorphanol 4 Theophylline 4 Thiopentone 4 Xylazine 4 Atropine 3 Phenobarbitone 3 Medetomidine 2

10 Metoclopramide 2 Adrenaline 1 Chlorpheniramine 1 Methadone 1 Methocarbamol 1 Morphine 1 Zolazepam/tiletamine 1 Table 5. Length of hospitalisation for 72 dogs exposed to Aplysia gigantea Duration of treatment Number of dogs Dead on arrival 2 Immediate discharge 22 <12 hours hours hours hours hours 1