August 2011 Technology and Welfare

Safe laboratory management for of medical importance

SARAH REED, MIKAELLA LOCK and STEVEN TRIM*

Venomtech Ltd, PO Box 468, Ramsgate, Kent CT11 1BD

*Corresponding author [email protected]

(Presented at Institute of Animal Technology Congress 2011)

Summary understanding of the ’ “normal” behaviour and Safe animal management is key to any facility; this is frequent, careful observation to identify any deviation especially true of those housing of significant from that normal behaviour. In addition, the husbandry medical importance. Procedures detailed here of arachnids of medical importance poses more demonstrate how dangerous arachnids including significant challenges in containment and safe handling (Family Buthidae ), wandering (Family (for both animal and technician) due to the size, speed, Ctenidae ), recluse spiders (Family Sicariidae ) and the aggression and venomous characteristics of many family containing the widow spiders can be species. safely managed in the laboratory environment. This dramatically reduces the risk of during This paper describes a method of performing routine husbandry and experimental procedures. Risks are husbandry by which risk of envenomation is reduced with modified enclosures that allow separation significantly reduced along with risk of escape or injury from the animals during most husbandry procedures of the animals and was originally presented as a poster and use of anaesthesia where contact is inevitable. at the Laboratory Animal Science Association (LASA) The equipment and techniques presented here thus Winter Meeting and an oral presentation at the Institute allow for greatly improved safety when working with of Animal Technology (IAT) Congress 2011. dangerous invertebrates. Medical importance Introduction Several species of Araneomorpha spiders produce Unlike most laboratory animal species, invertebrates, of significant medical importance, such as the such as spiders, are not protected by UK law. However, armed spiders of South America (Family Ctenidae, there are still compelling reasons for refining the species fera (Perty, 1833) and P. nigriventer husbandry of such animals, particularly as these (Keyserling 1897)), brown recluse spiders (Family species are of increasing interest as a source of novel Sicariidae , species Loxoceles laeta (Gertsch, 1961; therapeutics. Good husbandry will increase the non Nicolet, 1849 )) and the Theridiidae family lifespan and breeding potential which may have members – mactans (Fabricius, 1775) and financial implications and it is recognised that healthy L. hasselti (Thorell, 1870 ). Envenomation from animals, given the opportunity to exhibit “normal” arachnids of these genera have long been known to behaviours, make more valid experimental subjects. cause significant health effects in humans and are Possibly even more importantly for professional animal thus considered medically important. Of the species technologists is the principle that we have a duty of maintained within the Venomtech facilities the widows, care to any living creature held in captivity and good such as the black widow ( ) and the practice demands that we do our best to ensure each redback ( Latrodectus hasselti ), are the most creature remains, at the very least, healthy and likely to produce fatal envenomation, despite the fact comfortable. In the case of venomous invertebrates, that according to LD 50 studies their venom is of lower there is another important reason for cultivating good potency than that of other arachnids discussed herein, 1 husbandry practice; understanding behaviours and LD 50 1.34mg/kg . The widow’s neurotoxic venom leads methods of safe handling of venomous species could to immediate intense pain due to the stimulation of have serious health implications to the handler. The neurotransmittor release through increased exocytosis, basic principles of good animal husbandry are the from nociceptive (pain) synapses but this effect is not same for all species; maintenance of appropriate, specific to the nociceptive neurones and leads to hygienic environmental conditions including provision of muscle cramps, convulsions, vomiting, perfuse appropriate quantities of good quality food, a good sweating and occasionally death. Prior to the

85 Safe laboratory management for arachnids of medical importance development of an effective antivenin approximately medical attention as many other Buthidae scorpions do 6% of bites were fatal, this dropped to 0.2% with have a higher fatality rate. effective diagnosis and treatment 11 . The true role of effective diagnosis was emphasised recently when an The pharmacological potential of these is of otherwise healthy female died from an undiagnosed great interest as a source of biological tools and suspected to be Latrodectus mactans 2. To therefore present a need for Venomtech staff to reduce this potential it is vital that medical maintain these animals in captivity. Captive husbandry professionals know where dangerous spiders are being of these species presents a serious risk to the health kept outside of their natural range such that diagnosis and safety of staff, visitors, nearby businesses and the is swift. Brown recluse spiders ( Loxosceles species) public, risks which are greatly reduced by this are a source of significant morbidity throughout the husbandry protocol. The use of carbon dioxide (CO 2) for tropics and sub-tropical regions, due to the fact that anaesthesia in arachnids has been documented they are common in human dwellings and their bite several times but the integral use of anaesthesia as often goes unnoticed and therefore treatment is part of the husbandry program has not been described delayed due to lack of diagnosis. Although the mortality previously to the best of our knowledge. rate is low at 0.03 %3 the high morbidity is due to the high level of cytotoxic , such as Methods phospholipase, hyaluronidase and sphingomyelinase, All procedures were performed in accordance with the that breakdown the skin and muscle leaving large ethos of the Animals (Scientific Procedures) Act , 1986, ulcers that are difficult to treat effectively. In Paraná even though the arachnids are not protected species. state, , 50 % of Loxosceles bites are classified as All animals were obtained from private breeders at the moderate (characteristic lesion, erythrema and Terraristika expo , Hamm, Germany , March 2010. petechiae ) and 2% severe (with moderate symptoms but with added haemolysis) 3 so they have great Enclosures – The substrate is a 1-5 cm thick covering potential to impact the quality of life of victims and of vermiculite (Peregrine Livefoods Ltd) moistened therefore require specialist husbandry techniques to according to the humidity needs for the species being minimise this risk. housed, as suggested for laboratory husbandry of 9. Water is provided via a 5 cm diameter With venom nearly twice as potent as that of the black plastic water bowl (Vanishing World). widows 4, the armed spiders, often called wandering spiders ( Phoneutria species), are , unexpectedly, rarely Accommodation for small species (average fatal. These spiders are large, fast, aggressive and body length 2.97 cm +/- 0.48 cm) are standard 0.7 have the ability to produce frequent systemic litre secure locking plastic boxes (Really Useful envenomation but rarely do, in fact only in 0.5 % of Products Ltd) with eight , 3 mm holes along each side cases. 5 This could be an example of venom dose for ventilation. Larger (4cm body length) Parabuthus metering by spiders as has been observed in liosoma (Ehrenberg, 1828) are housed as above but in Parabuthus scorpions. 6 This is possibly because venom 1.7 litre flat Really Useful boxes (Really Useful Products is likely to be energetically expensive to produce, so Ltd). Scorpion body lengths are measured from large doses are only delivered in life threatening to the terminal wide segment of the circumstances, the arachnids preferring to warn . A shallow dish is provided for water and potential predators with threat displays, dry bites (those without envenomation) and low dose bites .

Although all scorpions produce feelings of fear and anxiety in many people, only a few are of significant medical importance and therefore listed on the Dangerous Wild Animals Act, 1976. Scorpions of the Buthidae family such as Leiurus quinquestriatus (Ehrenberg, 1828) , Parabuthus liosoma (Ehrenberg, 1828 ) and Androctonus australis (Linnaeus, 1758 ) are the main species capable of fatal envenomation 8 and therefore listed. Despite the fact that venom from the death stalker Leiurus quinquestriatus is nearly five times more potent in subcutaneous LD 50 studies with white mice 9 than that of the black widow Latrodectus mactans , it is responsible for very few fatalities. This is Figure 1. Modified Really Useful box for housing another potential example of dose metering 6 or could spiders of medical importance, showing feeding be due to a low venom yield. Even so, scorpion aperture in the lid (with closure bolt) and CO 2 line for envenomation is a serious event and may often require anaesthesia.

86 Safe laboratory management for arachnids of medical importance small sections of UPVC roundline guttering (Wickes Ltd) box is no longer detectable and when the spider can be are used to replicate natural crevices and burrows. inverted by tipping up the enclosure. This abolishment of the natural right-reflex is a firm measure of sufficient Typical containment for true spiders, many of which are anaesthesia depth. Time to anaesthesia is recorded listed in the Dangerous Wild Animals Act 1976, as and at this point the CO 2 supply is stopped. The amended 2010 (e.g. Loxosceles sp .), is made from a anaesthetised spider is then placed straight into a 2.1 litre plastic box with secure fitting lid (Really Useful clean enclosure and allowed to recover in fresh air. Products Ltd) (Figure 1). For larger spiders (greater than Should a spare, clean enclosure not be available then 5 cm conscious leg -span) the boxes are scaled up the spider can be placed into a 670 ml plastic food box, accordingly so most large Phoneutria species are (which is also used as an anaesthesia chamber ), housed in 3 litre enclosures. All Phoneutria species are ventral side up, in fresh air as most spiders will recover kept at ~80 % relative humidity while the Loxosceles before the enclosure can be fully cleaned. The time to and Latrodectids are kept much drier (~30 % relative recovery is recorded as the time taken for the right humidity) . A 10 mm diameter aperture for feeding reflex to return. Only when the spider is secure can the (crickets and flies) and topping up the water bowl is home cage be cleaned. Lab CO 2 levels are monitored drilled into the lid and secured with an M10 16 mm with a CO 2 Dräger Pac 7000 (Fisher Scientific) to nylon bolt (Nylons and Alloys Ltd). ensure compliance with the UK occupational exposure limit of 5000 ppm over 8 hours or 15000 ppm in any The individual enclosures are stacked in a glass 15 minute period. Another reason that Really Useful fronted shelving unit that is back heated (according to boxes are appropriate is that they are autoclavable, species, typically 27 +/- 3 °C) with a 100 W heat cable and resilient to chemical sterilisation such as 70 % (Eurorep Ltd) and thermostatically controlled with a isopropanol wipes (Hygiene for Less Ltd) and 2% Virkon Thermocontrol Prostat II (Lucky Reptile). This produces (Fisher Scientific). The time for the spider to right itself a thermal gradient that the animals use to naturally is recorded as this gives the most accurate ‘end-point’ thermoregulate. All enclosures are labelled with measure for full recovery. Most spiders will recover species name and ID code and similar species are kept before the enclosure has been cleaned so when the together. Velcro backed laminated labels on the front of clean enclosure is ready, anaesthesia is repeated and the glass cabinet display details of species (scientific the anaesthetised spider replaced into the clean and common names) complete with antivenin codes enclosure and monitored to confirm recovery. (where appropriate); these are to be removed and taken to hospital with the casualty should Due to the scorpions ’ lack of agility and inability to envenomation occur (see emergency procedure below) . climb vertical plastic surfaces they can be easily Water and humidity levels are checked regularly and manipulated by carefully, but firmly, grasping the last animals are fed feeder insects every 1-3 weeks as few segments of the metasoma, just anterior to the appropriate for the size and species. Spiders are fed telson, using a pair of 30 cm rubber tipped forceps prey insects just smaller than their opisthosoma, young (Cornish Crispa Company Ltd). The animal can then be spiders (up to 4 mm leg-span) are fed one fruitfly securely lifted swiftly into a clean enclosure. This (flightless mutant) (Livefoods system also attends to the animals’ welfare needs as Direct Ltd) every week, all other arachnids are fed the majority of predatory arachnids are ambush appropriate sized crickets or locusts ( Gryllus species hunters and therefore do not need large enclosures to and Schistocerca gregaria ) (Concrete Jungle Ltd) exercise in, as long as there is provision of shelter and weekly. Removal (spot cleaning) of discarded food or material to attach silk to build a retreat with. boluses and uneaten food is performed, after the spider is anaesthetised, as below. With the scorpion Alongside the safe husbandry methods detailed above enclosures, discarded food boluses and uneaten food specific emergency procedures are in place to reduce can be removed with 30 cm forceps. the risk of severe pathology developing should an envenomation incident occur. This includes a no lone- A 5 mm diameter occluded tube, with pinprick holes for working with dangerous arachnids policy. All staff in the CO 2 anaesthesia and water spray (via syringe) is fitted building are trained in essential life support first aid as just above the substrate level. At the top of the well as the procedure detailed within. Further opposite side of the box are eight 3 mm ventilation notification is required for the emergency services such holes such that a rising concentration of carbon dioxide that the local hospital and ambulance service are (CO 2) can be achieved. CO 2 anaesthesia is achieved aware of the species kept and that includes the through connection of the occluded tube to a low range nearest specialist poison unit who manage the CO 2 flow meter (Harvard apparatus) opened to 0.5 available antivenins. Unlike envenomation from a wild litre/minute for 0.7 litre boxes and 1 litre/minute for all where the priority is to move away from the other enclosures. Spiders undergoing anaesthesia are animal , which is often killed to be taken for closely monitored and are only considered identification in the laboratory environment, anaesthetised when a response to firm tapping of the identification is known and the first action must be to

87 Safe laboratory management for arachnids of medical importance contain the animal as arachnids can re-envenomate neurotoxic mode of action of the venoms apnea is likely multiple times . It is therefore essential that they be without cardiac arrest and therefore artificial secured for the sake of the casualty and the emergency ventilation is required without cardiac massage if a responders. Scorpions can easily be returned to their palpable pulse is present. home enclosure, using the technique detailed above. Spiders have greater agility and speed, thus the best Results emergency capture device is the domed top of a 2 litre No adverse events ( or escapees) have plastic bottle, as this has a top for a handle and can be been identified within Venomtech Laboratories since its occluded with a small rigid plastic sheet . The entire incorporation in March 2010. This can be accredited to device can then be placed into a suitable anaesthesia these procedures and the associated risk assessment. chamber. All nearby staff must be verbally alerted to Also we have observed many natural behaviours, such the incident during attempts to secure the animal. If as normal ecdysis (shedding skin), feeding, mating and several people are available then emergency services breeding in these animals , suggestive of the fact that should be called as soon as possible giving details of they are not severely affected by the CO 2 anaesthesia the number of casualties and approximate ages. The and husbandry procedures in place. Detailing caller must explain that the casualty has been Loxosceles laeta breeding as an example of such bitten/stung by an exotic venomous spider or scorpion breeding behaviour; the parents were introduced by (give species), indicate the location of the bite/sting, placing their , 2.1 litre, enclosures side-by-side and clarify that the casualty may require antivenin (give opening the lids whilst contained in a 30 litre box with antivenin code from the cage label), and provide CO 2 line attached. On the 18th December 2010 mating address details. Once the animal is securely contained proceeded as expected with the male signaling to the then the procedure is divided into spiders and female by tapping the enclosure sides. After scorpions due to different treatment needs. The priority approaching cautiously, copulation was observed, with spider envenomation is to prevent worsening of followed by naturally rapid retreat of the male to avoid local symptoms and monitor for systemic exposure, . Both adults were anaesthetised and whereas scorpion post-envenomation treatment should returned to their home enclosures. The egg sac was focus on prevention of systemic exposure as the only laid on the 23rd January 2011 and the female was local effect expected is pain, and systemic anaesthetised in her enclosure such that the egg sac envenomation is likely. If only two people are present, could be safely removed. The eggs were incubated the casualty should be stabilised first by applying a cold separately in a 125 ml clear polypropylene containers compress and elevating the bitten limb. The emergency (Fisher Scientific) at 24˚C and six spiderlings hatched services can then be called. It is essential that the on the 6th March. After a few days they were casualty is immobilised as quickly as possible as any anaesthetised and separated out into individual pots delay can increase the potential onset of systemic with a plastic leaf for a hide. As young spiderlings they symptoms. were fed flightless fruitflies ( Drosophila melanogaster ) and occasional small crickets ( Gryllodes sigillatus ) For scorpions the casualty should be laid down in a essentially following Donald Lowries’ suggested semi-recumbent position and the affected limb method 12 . bandaged from just below the site of envenomation up to the trunk in such a way as you would treat a sprain. The results presented below (Figure 2) show the This compression bandage is to limit the spread of the anaesthesia parameters for induction and recovery of venom into the blood stream via the lymphatic system the species used when anaesthetised in their home and is much the same as for a venomous snake bite 10 . enclosures. For the majority of individuals the time to It is critical that the casualty remains calm although it induction is not significantly different to the recovery seems antagonistic to the natural response it is time ( Phoneutria sp n=4 animals with 23 anaesthesias, important and is best achieved through training and p=0.94, and Loxosceles n=7 animals with 43 practice of emergency drills. The University of Adelaide anaesthesias, p=0.89). In contrast to this the has an excellent resource for envenomation and states Latrodectids statistically differ from this pattern as the many victims will be terrified, fearing sudden death induction time is significantly greater than the recovery and, in this mood, they may behave irrationally or even time (p=0.0005, Students T Test, n=7 animals, 11 hysterically. The basis for reassurance is the fact that anaesthesias). The outlier, , most venomous bites do not result in envenoming and displays the largest difference between induction and the effectiveness of modern medical treatment greatly recovery (p=0.03, n=1 animal, 4 anaesthesias). reduces the long-term effects 11 . At this stage the Occasionally times outside these may be recorded. In casualty’s condition should be frequently monitored our experience when there is a significant delay in time and any change in responsiveness, signs or symptoms taken to induce narcosis, this is usually due to the should be noted. If the casualty loses consciousness animal going into pre-ecdysis. The potential reason for and/or stops breathing then standard first aid this is the development of the new exoskeleton and procedures should be put in place, although due to the book lungs underneath the current cuticle as this is

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suspected to reduce the diffusion of CO 2 into the a comparison would be performed between routine tissues. Unusually long recovery times have been husbandry with and without the use of CO 2 observed on occasions with each species, often anaesthesia, but the authors feel the risk of greater than 600 seconds. The cause of this envenomation or escape are too great to warrant a phenomenon is unclear but further research is clearly trial. There have been anecdotal reports that the use of needed. CO 2 anaesthesia may shorten the lifespan of spiders in captivity 13 but this has not been observed in this study, potentially due to the use of a rising concentration of CO 2 and careful observation to prevent over anaesthesia. Although CO 2 is a very cost effective anaethesthetic agent these procedures could potentially be used with volatile anaesthetics such as isoflurane with minor modifications such as placing the enclosures inside a scavenged anaesthesia chamber and monitoring of residual isoflurane. Acknowledgements The author’s thanks go to Peter Gibbens (Concrete Jungle Ltd) for providing the premises for this work to take place and administering the license to work under Figure 2. Summary of anaesthesia data for the dangerous Wild Animals Act 1976, Stuart Thomson Araneomorph spiders, using their home enclosures, (PhotographyIs.com) for the photo of the enclosure. data averaged for genera except Phoneutria nigriventer Thomas Swan (Harvard Apparatus Ltd) for personal due to this species being substantially larger, than the communication around the use of our enclosures with other members of the currently housed. Error isoflurane and Dr Carol Trim for critical review of the bars show standard deviation of full data. The manuscript before submission. difference between induction and recovery times for Phoneutria nigriventer and Latrodectius sp. are References significant (p=0.03 and p=0.0005 respectively). 21 Rauber, A . (1984). Black Widow Spider Bites. Journal of Toxicology : Clinical Toxicology Vol. 21 No. 44, 473-485. Discussion 22 Gaisford, K . and Kautz, D.D. (2011). Black Widow Spider The authors believe these procedures allow dangerous Bite a case study. Dimensions of Critical Care Nursing arachnids to be maintained in a laboratory environment Vol. 30(2), 79-86. 23 Marques da Silva, E., Souza-Santos, R., Fischer, M.L. in a safe and effective manner, thus, attending to the and Rubio, G.B.G. (2006) . Loxosceles spider bites in the welfare needs of the species at the same time as state of Parana, Brazil: 1993-2000. Journal of Venomous providing a physical barrier between the staff and the Animals and Toxins including Tropical Diseases , Vol. 12, animals for the majority of routine procedures and No.1, p.110-123. utilising anaesthesia for those tasks, such as 24 Bucherl, W. (1969). Most Important South American enclosure cleaning, where contact is unavoidable. Spiders of the Genera Phoneutria, Loxosceles, Lycosa, Many aspects of natural behaviour have frequently and Latrodectus. American Zoologist , Vol. 9. 157-159. been observed in these species being housed in this 25 Bucaretchi, F. Deus Reinaldo CR. Hyslop S, Roberto P. manner, including breeding of a few species. The CO (2000). A clinico-epidemiological study of bites by 2 spiders of the genus Phoneutria. Revista do Instituto de anaesthesia data presented here shows that the Medicina Tropical de São Paulo Vol. 42 (1): 17-21. majority of species used in this study have similar 26 Nisani, Z. and Hayes, W.K. (2011). Defensive stinging by induction and recovery times; this allows for prediction Parabuthus transvaalicus scorpions: risk assessment of recovery time in the majority of Phoneutria and and venom metering. Animal Behaviour . Vol. 81, 627-633. Loxoceles species, thus predicting the safe working 27 Keegan, H.L. (1980). Scorpions of medical importance. time. The Latrodectus species are an exception to this University Press of Mississippi. with a recovery time half that of the induction time. This 28 Mullen, G. and Durden, L. (2009). Medical and could potentially be due to the enclosures although this Veterinary Entomology. Academic Press, Massachusetts . 29 phenomenon still exists when using a 670 ml Gershenz, L .S. (1996). Laboratory culture techniques for anaesthesia chamber so it is more likely to be due to the goliath (Theraphosa blondi) (Latreille, 1804) and the Mexican redknee tarantula (Brachypelma physiological differences. Further work is required in smithi) (Araneae: Theraphosidae). American Zoo and this along and with the single large Phoneutria Aquarium Association Regional Conference Proceedings: nigiriventer as although the multiple anaesthesias 773-777 . show a difference between induction and recovery, 10 Warrell, D . (2005). Treatment of bites by adders and studies have only been conducted on one animal due exotic venomous snakes. British Medical Journal 331: to the difficulty obtaining this species. In an ideal state 1244.

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11 University of Adelaide clinical toxinology resource (www.toxinology.com/fusebox.cfm?fuseaction=main.first _aid.firstaid&id=FAD-04) 12 Lowrie, D.C. (1987). Effects of diet on the development of Loxosceles laeta (Nicolet) (Araneae, Loxoscelidae). Journal of Vol. 15, 303-308. 13 Lewbart, G.A. (2006). Invertebrate Medicine. Blackwell Publishing Ltd, Oxford.

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