hygiene protocol for the control of disease in frogs (pdf
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NSWNATIONALPARKS ANDWILDLIFESERVICE
Threatened Species ManagementInformation Circular No. 6
August 2001
hygiene protocol for thecontrol of disease in
frogs
© NSW National Parks and Wildlife Service, 2001.
This work is copyright. However, material presented in thisprotocol may be copied for personal use or utilised formanagement and educational purposes, providing that anyextracts are fully acknowledged. Apart from this and any otheruse as permitted under the Copyright Act 1968, no part may bereproduced without prior written permission from NPWS.
NSW National Parks and Wildlife Service43 Bridge Street(PO Box 1967)Hurstville NSW 2220Ph 1300 36 1967www.npws.nsw.gov.au
Requests for information are best directed to:
NSW National Parks and Wildlife ServiceDeclining Frog Working Group CoordinatorBiodiversity Management UnitPO Box 1967Hurstville NSW 2220Ph (02) 9585 6426
This document can be sourced from the NPWS website:www.npws.nsw.gov.au/wildlife/licence/frog.html
This document should be cited as:NSW National Parks and Wildlife Service (2001). Hygieneprotocol for the control of disease in frogs. Information CircularNumber 6. NSW NPWS, Hurstville NSW.
ISBN 0 7313 6372 8
Acknowledgments
NSW National Parks and Wildlife Service Declining FrogWorking Group who recommended the preparation and providedinput into the development of this strategy.
Ross Wellington and Ron Haering (both NPWS) the authors ofthis document.
Thanks to Jack Baker, Lee Berger, Mark Endersby, Jeff Hardy,Frances Hulst, Alex Hyatt, Keith McDougall, Diana Mendez,Deborah Pergolotti, Graham Pyke, Marjo Rauhala, JulieRavallion, Karrie Rose, Lothar Voigt and Arthur White for theiradvice and/or technical review.
This hygiene protocol is an adaptation of the DecliningAmphibian Population Task Force (DAPTF) Fieldwork Code ofPractice and the recommendations of Speare et al. (1999) andhas drawn on recommendations from earlier guidelines preparedby Environment ACT.
Foundation for National Parks and Wildlife funded the printingof this protocol.
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1. INTRODUCTION......................................................................................... 1
1.1 WHO SHOULD READ THIS DOCUMENT? ......................................................................1
1.2 BACKGROUND ........................................................................................................................1
1.2.1 Amphibian Chytrid Fungus.............................................................................................1
1.3 OBJECTIVES ...............................................................................................................................2
2. SITE HYGIENE MANAGEMENT ................................................................ 3
2.1 DEFINING A SITE ......................................................................................................................3
2.2 ON-SITE HYGIENE ...................................................................................................................3
2.3 HANDLING OF FROGS IN THE FIELD ...............................................................................4
2.4 DISINFECTION METHODS ...................................................................................................5
3. CAPTIVE FROG HYGIENE MANAGEMENT ........................................... 6
3.1 HOUSING FROGS AND TADPOLES ....................................................................................6
3.2 TADPOLE TREATMENT...........................................................................................................6
3.3 FROG TREATMENT ..................................................................................................................6
3.4 DISPLACED FROGS ..................................................................................................................7
3.4.1 Banana Box Frogs ............................................................................................................8
3.4.2 Cane Toads .......................................................................................................................8
3.4.3 Local Frog Species...........................................................................................................8
4. SICK OR DEAD FROGS .............................................................................. 9
4.1 SYMPTOMS OF SICK AND DYING FROGS .......................................................................9
4.2 WHAT TO DO WITH SICK OR DEAD FROGS.............................................................. 10
Appendix 1 HYGIENE PROTOCOL CHECKLIST AND FIELD KIT ................................ 12
Appendix 2 DESIGNATED SICK AND DEAD FROG RECIPIENTS ................................ 13
Appendix 3 NSW ANIMAL WELFARE ADVISORY COUNCIL METHODOLOGY ..... 14
Appendix 4 LICENSED WILDLIFE CARER AND RESCUE ORGANISATIONS .......... 15
Appendix 5 SICK OR DEAD FROG COLLECTION FORM ............................................. 16
hygiene protocol forthe control of disease in
frogs
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1 introduction
1.1 Who should read thisdocument?
This protocol is intended for use by allresearchers, wildlife consultants, faunasurveyors and students undertaking frogfield-work. In addition, the protocolshould be read by National Parks andWildlife Service (NPWS) personnel, frogkeepers, wildlife rescue and carerorganisations, herpetological/frog interestgroups/societies, fauna park/zoo operators/workers and other individuals whoregularly deal with or are likely toencounter frogs.
This protocol outlines the expectations ofthe NPWS regarding precautionaryprocedures to be employed when workingwith frog populations. The intention is topromote implementation of hygieneprocedures by all individuals working withfrogs. New licences and licence renewalswill be conditional upon incorporation ofthe protocol. The NPWS recognises thatsome variation from the protocol may beappropriate for particular research and froghandling activities. Such variationproposals should accompany any licenceapplication or renewal to the NPWS.
1.2 Background
1.2.1 Amphibian Chytrid Fungus
The apparent decline of frogs, includingextinctions of species and localpopulations, has attracted increasedinternational and national concern. Many
potential causes for frog declines havebeen proposed (eg see Pechmann et al.,1991; Ferrero and Bergin, 1993; Pechmannand Wilbur, 1994; Pounds and Crump,1994; Pounds et al., 1997). However, thepatterns of decline at many locationssuggest that epidemic disease maybe thecause (Richards et al., 1993; Laurance etal., 1996; Alford and Richards, 1997).Recent research has implicated a water-borne fungal pathogen Batrachochytriumdendrobatidis as the likely specific causativeagent in many of these declines both inAustralia and elsewhere (Berger et al.,1998; 1999). This agent is commonlyknown as the amphibian or frog chytridfungus and is responsible for the diseaseChytridiomycosis (Berger et al., 1999).
B. dendrobatidis is a form of fungusbelonging to the phylum Chytridiomycota.Most species within this phylum occur asfree-living saprophytic fungi in water andsoil and have been found in almost everytype of environment including deserts,arctic tundra and rainforest and areconsidered important primary biodegraders(Powell 1993). B. dendrobatidis is a uniqueparasitic form of Chytridiomycete fungi, inthat it invades the skin of amphibians,including tadpoles, often causing sporadicdeaths with up to 100% mortality in somepopulations. Chytridiomycosis has beendetected in over 40 species of nativeamphibian in Australia (Mahony andWekman 2000). However, it is notcurrently known whether the fungus isendemic or exotic to Australia.
This information circular outlines measures to:
• Prevent or reduce disease causing pathogens being transferred within and between
wild populations of frogs.
• Ensure captive frogs are not infected prior to release.
• Deal safely with unintentionally transported frogs.
• Assist with the proper identification and management of sick and dead frogs in the wild.
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The infective stage of B. dendrobatidis isthe zoospore and transmission requireswater (Berger et al.,1999). Zoosporesreleased from an infected amphibian canpotentially infect other amphibians in thesame water. More research is needed onthe dynamics of infection in the wild.B. dendrobatidis is known to be susceptibleto seasonal temperature changes,dehydration, salinity, water pH, light,nutrition and dissolved oxygen(Berger et al., 1999).
1.3 Objectives
The objectives of the hygiene protocol areto:• Recommend best-practice procedures
for NPWS personnel, researchers,consultants and other frog enthusiastsor individuals who handle frogs.
Life cycle of frog chytrid fungus from infective free-living zoospore stage to sporangium (adapted fromL. Berger).
• Suggest workable strategies for thoseregularly working in the field with frogsor conducting fieldwork activities inwetlands and other aquaticenvironments where there is thepotential for spreading pathogens suchas the frog chytrid fungus.
• Provide background information andguidance to people who provide adviceor supervise frog related activities.
• Provide standard licence conditions forworkers engaged in frog relatedactivities.
• Inform Animal Care and EthicsCommittees (ACEC) for theirconsideration when granting researchapprovals.
free-living zoosporesporangium
2 site hygiene management
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When working along a river or stream oraround a wetland or a series ofinterconnecting ponds it is reasonable, inmost instances, to treat such examples as asingle site for the purposes of this protocol.Such a case would occur in areas wherefrogs are known to have free interchangebetween ponds.
Where a stream consists of a series ofdistinctive tributaries or sub-catchments orwhere there is an obvious break or divisionthen they should be treated as separatesites, particularly if there is no knowninterchange of frogs between sites.
2.2 On-site hygiene
When travelling from site to site it isrecommended that the following hygieneprecautions be undertaken to minimise thetransfer of disease from footwear,equipment and/or vehicles.
Footwear
Footwear must be thoroughlycleaned and disinfected at thecommencement of fieldwork andbetween each sampling site.
This can be achieved by initially scrapingboots clear of mud and standing the soles ina disinfecting solution. The remainder ofthe boot should be rinsed or sprayed with adisinfecting solution that containsbenzalkonium chloride as the activeingredient. Disinfecting solutions should beprevented from entering any water bodies.
Rubber boots such as ‘gum boots’ or‘Wellingtons’ are recommended because ofthe ease with which they can be cleanedand disinfected.
Several changes of footwear baggedbetween sites might be a practicalalternative to cleaning.
A checklist of riskmanagementprocedures andrecommendedstandard hygiene kitis provided inAppendix 1. Pleasenote Footnote 1 onpage 4.
Individuals studying frogs often travel andcollect samples of frogs from multiple sites.Some frog populations can be particularlysensitive to the introduction of infectiouspathogens such as the frog chytrid fungus.Also, the arrangement of populations inthe landscape may make frogs particularlyvulnerable to transmission of infectiouspathogens. Therefore, it is important thatfrog workers recognise the boundariesbetween sites and undertake measureswhich reduce the likelihood of spreadinginfection.
Where critically endangered species orpopulations of particular risk are known tooccur, this protocol should be applied oververy short distances ie a single site mayneed to be subdivided and treated asseparate sites.
When planning to survey multiple sites,always start at a site where frog chytridfungus is not known to be present beforeentering other infected areas.
2.1 Defining a site
Defining the boundary of a site maybeproblematic. In some places, the boundarybetween sites will be obvious but in others,less so. Undertaking work at a number ofsites or conducting routine monitoring at aseries of sites within walking distancecreates obvious difficulties with boundarydefinitions. It is likely that defining theboundary between sites will differ amonglocalities. It may be that a natural orconstructed feature forms a logicalindicator of a site boundary eg a road/track, a large body of water such as a riveror the sea, a marked habitat change or acatchment boundary.
As a guiding principle, eachindividual waterbody should beconsidered a separate site.
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Equipment
Equipment such as nets, balances,callipers, bags, scalpels, headlamps,torches, wetsuits and waders etcthat are used at one site must becleaned and disinfected before re-use at another site.
Disposable items should be used wherepossible. Non-disposable equipmentshould be used only once during aparticular field exercise and disinfectedlater or disinfected at the site between usesusing procedures outlined in 2.4 below.
Vehicles
Where necessary, vehicle tyresshould be sprayed/flushed with adisinfecting solution in high-riskareas.
Transmission of disease from vehicles isunlikely to be a problem. However, if avehicle is used to traverse a known frogsite, which could result in mud and waterbeing transferred to other bodies of wateror frog sites, then wheels and tyres shouldundergo cleaning and disinfection. Thisshould be carried out at a safe distancefrom water bodies, so that the disinfectingsolution can infiltrate soil rather than run-off into a nearby water body.
Spraying with ‘toilet duck’ (activeingredient benzalkonium chloride) isrecommended to disinfect car wheelsand tyres.
Cleaning of footwear before getting backinto the car will prevent the transfer ofpathogens from/to vehicle floor andcontrol pedals.
2.3 Handling of frogs in the field
The spread of pathogenic organisms, suchas the frog chytrid fungus, may occur as aresult of handling frogs.
Frogs should only be handled whennecessary.
Where handling of frogs is necessary therisk of pathogen transfer should beminimised as follows:• Hands should be either cleaned and
disinfected between samples or a newpair of disposable gloves used for eachsample1. This may be achieved bycommencing with a work area that hasa dish containing a disinfectingsolution and paper towels.
• A ‘one bag – one frog’ approach to froghandling should be used especiallywhere several people are workingtogether with one person processingfrogs and others doing the collecting.Bags should not be reused.
• A ‘one bag – one sample’ approach totadpole sampling should be used. Bagsshould not be reused.
Researchers who use toe clipping orPassive Integrated Transponder (PIT)tagging are likely to increase the risk oftransmitting disease between frogs due tothe possibility of directly introducingpathogens into the frogs’ system. This canbe minimised by using:• Disposable sterile instruments• Instruments disinfected previously and
used once
• Instruments disinfected in betweeneach frog
1 As a principle, this protocol assumes that not all frogs in an infected pond will be contaminated by the frogchytrid fungus. The infective load of a body of water may not be high enough to cause cross contamination ofindividual frogs in the same pond. Therefore care should be taken to use separate gloves and bags and cleanhands for each sample, to avoid transmission of high infective loads between individuals.
Disinfectingsolutions containingbenzalkoniumchloride are readilyavailable from localsupermarkets.Some brandsinclude Toilet Duck,Sanpic, New Clenzand Pine Clean.
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Open wounds from toe clipping and PITtagging should be sealed with acyanoacrylate compound such as Vetbond©to reduce the likelihood of entry ofpathogens. The NPWS ACEC furtherrecommends the application of topicalanaesthetic Xylocaine© cream andBetadine© disinfectant (1% solution)before and after any surgical procedure.This should then be followed by thewound sealant.
All used disinfecting solutions, gloves andother disposable items should be stored ina sharps or other waste container anddisposed or sterilised appropriately at thecompletion of fieldwork. Disinfectingsolutions must not come into contact withfrogs or be permitted to contaminate anywater bodies
2.4 Disinfection Methods
Disinfecting agents for hands andequipment must be effective againstbacteria and both the vegetative and sporestages of fungi. The following agents arerecommended:• Chloramine and Chlorhexidine based
products such as Halamid©, Halasept©
or Hexifoam© are effective against bothbacteria and fungi. These products aresuitable for use on hands, footwear,instruments and other equipment. Themanufacturers instructions should befollowed when preparing thesesolutions.
• Bleach and alcohol (ethanol ormethanol), diluted to appropriateconcentrations can be effective againstbacteria and fungi. However, thesesubstances may be less practical becauseof their corrosive and hazardous nature.When using methanol either:• immerse in 70% methanol for 30
minutes or
• dip in 100% methanol then flamefor 10 seconds or boil in water for 10minutes
Fresh bleach (5% concentration) may bealso effective against other frog pathogenssuch as Rana Virus.
Some equipment not easily disinfected inthese ways can be effectively cleaned usingmedical standard 70% isopropyl alcoholwipes – Isowipes©.
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3 captive frog hygiene management3.1 Housing frogs and tadpoles
Frogs and tadpoles should only beremoved from a site whenabsolutely necessary.
When it is necessary for frogs or tadpolesto be collected and held for a period oftime, the following measures should beundertaken:• Animals obtained at different sites
should be kept isolated from each otherand from other captive animals.
• Aquaria set up to hold frogs should notshare water, equipment or any filtrationsystem. Splashes of water fromadjacent enclosures or drops of wateron nets may transfer pathogensbetween enclosures.
• Prior to housing frogs or tadpoles,ensure that tanks, aquaria and anyassociated equipment are disinfected.
• Tanks and equipment should becleaned, disinfected and driedimmediately after frogs/tadpoles areremoved.
3.2 Tadpole treatment
In most instances:
Release to the wild of tadpolesheld or bred in captivity shouldbe avoided.
When contemplating a release of captivebred tadpoles for conservation purposes aTranslocation Proposal should besubmitted to the NPWS and pathologicalscreening for disease should be undertaken(see also NPWS Draft TranslocationPolicy). Tadpoles can be tested byrandomly removing 10 individuals at 6weeks and again at 2 weeks beforeanticipated release. Testing could beundertaken by the pathology section atTaronga Zoo, Newcastle University,CSIRO Australian Animal HealthLaboratories at Geelong and James CookUniversity at Townsville. Such anarrangement would need to be negotiatedby contacting one of these institutionswell before the anticipated release date.(see Appendix 2 for contact details)
The NPWS will licence the NSWDepartment of Education and Training(DET) to allow school students and/orteachers to remove tadpoles for classroomlife cycle studies. They will be authorisedto remove a maximum of 20 individualsfrom only one location and each schoolwill also require endorsement from DETAnimal Care and Ethics Committee andcomply with this protocol.
Tadpoles collected for these purposes areto be obtained from the local area of theschool and are not to be obtained fromNPWS Reserves. As soon as tadpoleshave transformed, froglets must bereturned to the exact point of capture.Tadpoles from different locations are notto be mixed.
Antifungal cleansing treatments to cleartadpoles of the frog chytrid fungus arecurrently being trialed. In the future, sucha treatment may be an added procedurerequired prior to froglet releases.
Detailedinformation onsafely maintainingfrogs in captivity isprovided in Voigt(2001).
Careful maintenance of your enclosures will ensurea safe and hygienic environment for captive frogsand tadpoles.
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3.3 Frog treatment
The rigour with which frogs must betreated to ensure pathogens are notintroduced to native populations meansthat any proposal for the removal of adultfrogs (particularly threatened species) fromwild populations should be given carefulconsideration.
When it is essential for frogs to beremoved from the wild, the followingshould apply.
Individuals to be released should bequarantined for a period of 2 months andmonitored for any signs of illness ordisease.
Frogs must not be released if any evidenceof illness or infection is detected. If illnessis suspected, further advice must be soughtfrom a designated frog recipient(Appendix 2) as soon as possible todetermine the nature of the problem.Chytridiomycosis can be diagnosed in livefrogs by microscopical examination ofpreserved toe clips or from shedding skinsamples. Research is still in progress onthe development of a simple technique forthe detection of Chytridiomycosis and atreatment for infected frogs.
Current methods which may be usedinclude:
• A technique for the treatment ofpotentially infected frogs is to place thefrogs individually in a 1mg/Lbenzalkonium chloride solution for 1hour on days 1, 3, 5, 9, 11 and 13 of thetreatment period. Frogs are thenisolated/quarantined for two months.This and other possible treatments aredocumented in Berger and Speare(1998)
• Betadine© and Bactone© treatmentshave also been used on adult frogs withsome success (M. Mahony, NewcastleUniversity pers. comm.)
• Itraconazole© is an expensive drugwhich has been used successfully (LeeBerger CSIRO Australian AnimalHealth Laboratory pers. comm.).Information on this method is availableon the Website http://www.jcu.edu.au/school/PHTM/frogs/adms/attach6.pdf.
Frogs undergoing treatment should behoused individually and kept separate fromnon-infected individuals.
3.4 Displaced frogs
Displaced frogs are those native frogspecies and introduced Cane Toads (Bufomarinus) which have been unintentionallytransported around the country with freshproduce, transported produce andlandscaping supplies. Procedures to beundertaken when encounteringintroduced/displaced native frog species(as well as Cane Toads) are as follows.
3.4.1 Banana box frogs
‘Banana Box’ frog is the term used todescribe several native frog species (usuallyLitoria gracilenta, L. infrafrenata, L.bicolor and L. caerulea) commonlytransported in fruit and vegetableshipments and landscaping supplies. Inthe past, well meaning individuals haveattempted to return these frogs to theirplace of origin but this is usuallyimpossible to do accurately. There is risk ofspread of disease if these frogs aretransferred from place to place.
It is strongly recommended that:
Displaced Banana Box frogsshould be treated as if they areinfected and should not to befreighted anywhere for release tothe wild unless specifically approvedby NPWS.
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When encountering a displaced frog:• Contact a licensed wildlife carer
organisation to collect the animal. Thefrog should then undergo a quarantineperiod of 2 months along with anapproved disinfection treatment.
• Post-quarantine, the frog (if one of thespecies identified above) may betransferred to a licensed frog keeper.All other species require the permissionfrom NPWS Wildlife Licensing (WLU)prior to transfer. Licensed carer groupsare to record and receipt frogs obtainedand disposed of in this way.
• Licensed Frog Keepers are to list thesefrogs in their annual licence returns toNPWS.
Frogs held by licensed Frog Keepers arenot to be released to the wild except withspecific NPWS approval.
Displaced frogs may be made available torecognised institutions for researchprojects, display purposes or perhapsoffered to the Australian Museum asscientific specimens once approval hasbeen provided by the NPWS WLU.
3.4.2 Cane toads
Cane toads are known carriers ofthe Frog chytrid fungus and shouldnot be knowingly transported orreleased to the wild.
If a cane toad is discovered outside of itsnormal range, it should be humanelyeuthanased in accordance with therecommended NSW Animal WelfareAdvisory Council procedure (seeAppendix 3). Care should be taken toavoid euthanasia of native species due tomistaken identity.
3.4.3 Local frog species
Frogs encountered on roads, arounddwellings and gardens or inswimming pools should not beconsidered as displaced frogs.
Frogs encountered in these situationsshould be assisted off roads, away fromdwellings, or out of swimming poolspreferably to the nearest area of vegetationor suitable habitat.
Incidences of frogs spawning or tadpolesappearing in swimming pools shouldbe referred to a wildlife carer/rescueorganisation for assistance(see Appendix 4).
Contact the Frogwatch Helpline if you areunsure whether a frog is a local species ordisplaced.
An NPWSinformationbrochure titled‘Cane Toads inNSW’ providesfurther informationon cane toads andassistance withidentification ofsome of thecommonlymisidentified nativespecies. Thisinformation is alsoavailable on theNPWS website.
Frogs are often unintentionally transported withfresh produce and landscaping supplies. They arecollectively known as ‘banana box’ or displaced frogs.
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Unless an obvious cause of illness or deathis evident (eg predation or road mortality):Sick or dead frogs encountered in the wildshould be collected and disposed of inaccordance with the procedures describedin section 4.2 below.
4.1 Symptoms of sickand dying frogs
Sick and dying frogs exhibit a range ofsymptoms characteristic of chytridinfection. Symptoms may be expressed inthe external appearance or behaviour ofthe animal. A summary of these symptomsare described below. More detailedinformation can be found in Berger et al.,(1999) or at the James Cook UniversityAmphibian Disease website at:http://www/jcu.edu.au/school/phtm/PHTM/frogs/ampdis.htm. A directory ofother useful websites is provided inNPWS (2001).
Appearance(one or more symptoms)
• darker or blotchy upper (dorsal) surface• reddish/pink-tinged lower (ventral)
surface and/or legs and/or webbing ortoes
• swollen hind limbs• very thin or emaciated• skin lesions (sores, lumps)• infected eyes
• obvious asymmetric appearance
Behaviour (one or more symptoms)• lethargic limb movements, especially
hind limbs• abnormal behaviour (eg a nocturnal,
burrowing or arboreal frog sitting in theopen during the day and making novigorous attempt to escape whenapproached)
• little or no movement when touched
4 sick or dead frogs
Diagnostic behaviour tests
Sick frogs will fail one or more of the following tests:
test healthy sick
Gently touch with finger Frog will blink Frog will not blink above the eye
Turn frog on its back Frog will flip Frog will remain on its backback over
Hold frog gently by its Frog will use its No response from frogmouth forelimbs to try
to remove grip
Great barred frog (Mixophyes fasciolatus) with severeChytrid infection — note lethargic attitude andsloughing skin. Photo: L. Berger
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4.2 What to do with sick ordead frogs
A procedure for the preparation andtransport of a sick or dead frog is givenbelow2. Adherence to this procedure willensure the animal is maintained in asuitable condition for pathologicalexamination and assist the NPWS andresearchers to determine the extent of thedisease and the number of species affected.• Disposable gloves should be worn when
handling sick or dead frogs. Avoidhandling food and touching yourmouth or eyes as this could transferpathogens and toxic skin secretionsfrom some frog species.
• New gloves and a clean plastic bagshould be used for each frog specimento prevent cross-contamination. Whengloves are unavailable, use animplement to transfer the frog to acontainer rather than using bare hands.
• If the frog is dead, keep the specimencool and preserve as soon as possible (asfrogs decompose quickly after deathmaking examination difficult).Specimens can be fixed/preserved in70% ethanol or 10% buffered formalin.
Cut open the belly and place the frog inabout 10 times its own volume ofpreservative. Alternatively, specimens canbe frozen (although this makes tissuesunsuitable for some tests). If numerousfrogs are collected, some should bepreserved and some should be frozen.Portions of a dead frog can be sent foranalysis eg a preserved foot, leg or aportion of abdominal skin.
• The container should be labelledshowing at least the species, date andlocation. A standardised collectionform is provided in Appendix 5.
• If the frog is alive but unlikely tosurvive transportation (death appearsimminent), euthanase the frog (seeAppendix 3) and place the specimen ina freezer. Once frozen, the specimen isready for shipment to the addressprovided below.
• If the frog is alive and likely to survivetransportation, place the frog intoeither a moistened cloth bag with somedamp leaf litter or into a plastic bagwith damp leaf litter and partiallyinflated before sealing. Remember tokeep all frogs separated duringtransportation.
• Preserved samples can be sent in jars orwrapped in wet cloth, sealed in bagsand placed inside a padded box.
• Send frozen samples in an esky with dryice (available from BOC/CIG Gasoutlets).
• Place live or frozen specimens into asmall styrafoam esky (available from K-Mart/Big W for approximately $2.50).
• Seal esky with packaging tape andaddress to one of the laboratories listedin Appendix 4.
• Send the package by courier (NPWSuse TNT Express Overnight).
• Keep all receipts for esky, ice, coolerbricks and courier for reimbursementfrom NPWS Threatened Species UnitNorthern Directorate (Appendix 3).
2 The measures described below are standard procedures and may vary slightly depending on the distance andtime required to reach the intended recipient. Contact the intended recipient of the sick or dead frog prior tosending to confirm the appropriate procedure.
Further informationon sick and dyingfrogs is available onthe AmphibianDisease Home Pageathttp://www.jcu.edu.au/dept/PHTM/frogs/ampidis.htm — inparticular refer to‘What to do withdead or ill frogs’.
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National Parks and Wildlife Service(2000) Helping frogs survive- A guide forfrog enthusiasts. (Prepared by Voight, L.,Haering, R., and Wellington, R). NPWSHurstville. NSW.
Pechmann, J.H.K. and Wilbur, H.M.(1994) Putting declining amphibianpopulations into perspective: naturalfluctuations and human impacts.Herpetologica 50: 64-84.
Pechmann, J.H.K., Scott, D.E.,Semlitsch, R.D., Caldwell, J.P., Vitt, L.J.and Gibson, J.W. (1991) Decliningamphibian populations: the problem ofseparating human impacts from naturalfluctuations. Science 253: 892-895.
Pounds, J.A. and Crump, M.L. (1994)Amphibian declines and climatedisturbance: the case for the golden toadand harlequin frog. Conserv. Biol. 8: 72-85.
Pounds, J.A., Fogden, M.P.L., Savage,J.M. and Gorman, G.C. (1997) Test ofnull models for amphibian declines on atropical mountain. Conserv. Biol. 11:1307-1322.
Powell, M.J. (1993) Looking at mycologywith a Janus face: A glimpse ofchytridiomycetes active in theenvironment. Mycologia 85: 1-20.
Richards, S.J., McDonald, K.R. andAlford, R.A. (1993) Declines inpopulations of Australia’s endemic tropicalrainforest frogs. Pacific Conserv. Biol. 1:66-77.
Speare, R., Berger, L. and Hines, H.(1999) How to reduce the risk of youtransmitting an infectious agent betweenfrogs and between sites. AmphibianDiseases Home Page 22/1/99, (http://www.jcu.edu.au/dept/PHTM/frogs/ampdis.htm.).
Voight, L. (2001) Frogfacts No. 8. Froghygiene for captive frogs (draftpublication). FATS. Group. Sydney.
5 referencesAlford, R.A. and Richards, S.J. (1997)Lack of evidence for epidemic disease asan agent in the catastrophic decline ofAustralian rainforest frogs. Conserv. Biol.11: 1026-1029.
Berger, L., Speare, R. (1998)Chytridiomycosis - a new disease ofamphibians. ANZCCART News 11(4) : 1-3.
Berger, L., Speare, R., Daszac, P., Green,D.E., Cunningham, A.A., Goggin, C.L.,Slocombe, R., Ragan, M.A., Hyatt,A.D., McDonald, K.R., Hines, H.B.,Lips, K.R., Marantelli, G. and Parkes, H.(1998) Chytridiomycosis causesamphibian mortality associated withpopulation declines in the rainforests ofAustralia and Central America. Proc. Nat.Acad. Sci. 95: 9031-9036.
Berger, L., Speare, R. and Hyatt, A. (1999)Chytrid fungi and amphibian declines:Overview, implications and future directions.In: Campbell, A. (Editor) Declines anddisappearances of Australian frogs.Biodiversity Group, Environment Australia.
Environment ACT (1999) Guidelines forminimising introduction and spread of frogpathogens. Environment ACT. Canberra.
Ferrero, T.J. and Bergin, S. (1993)Review of environmental factorsinfluencing the declines of Australianfrogs. In: Lunney, D. and Ayers, D.(Editors) Herpetology in Australia: adiverse discipline. Trans. R. Zool. Soc.Mosman.
Laurance, W.F., McDonald, K.R. andSpeare, R. (1996) Epidemic disease andcatastrophic decline of Australian rainforestfrogs. Conserv. Biol. 77: 203-212.
Mahony, M. and Werkman, H. (2000)The distribution and prevalence ofChytrid fungus in frog populations ineastern New South Wales and developinga means to identify presence or absence ofChytrid fungus in the field. Unpublishedreport to NSW National Parks andWildlife Service.
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appendix 1hygiene protocol checklist and field kitThe following checklist and field kit are designed to assist with minimising the risk of
transferring pathogens between frogs.
Have you considered the following questions before handling frogs in the field:• Has your proposed field trip been sufficiently well planned to consider hygiene issues?
• Have you taken into account boundaries between sites (particularly where endangeredspecies or populations at risk are known to occur)?
• Have footwear disinfection procedures been considered and a strategy adopted?• Have you planned the equipment you will be using and developed a disinfection
strategy?• Are you are planning to visit sites where vehicle disinfection will be needed (consider
both vehicle wheels/tyres and control pedals) and if so, do you have a plan to deal withvehicle disinfection?
• Have handling procedures been planned to minimise the risk of frog to frog pathogentransmission?
• Do you have a planned disinfection procedure to deal with equipment, apparel anddirect contact with frogs?
If you answered NO to any of these questions please re-read the relevant sectionof the NPWS Hygiene Protocol for the Control of Disease in Frogs and apply asuitable strategy.
Field hygiene kit
When planning to survey frogs in the field a portable field hygiene kit should be assembledto assist with implementing this protocol. Recommended contents of a field hygiene kitwould include:• Small styrofoam eski• Disposable gloves• Disinfectant spray bottle (atomiser
spray) and/or wash bottle• Disinfecting solutions• Wash bottle• Scraper or scrubbing brush
• Small bucket• Plastic bags large and small• Container for waste disposal• Materials for dealing with sick and dead frogs (see section 4.2)
appendix 2
13
Contact one of the following specialists toarrange receipt and analyse sick and deadfrogs. Make contact prior to dispatchingpackage:
Karrie RosePathology Taronga ZooPO Box 20MOSMAN NSW 2088Phone: 02 9978 4749Fax: 02 9978 [email protected]
Diana MendezSchool of Public Healthand Tropical MedicineJCU House — Suite 1157 Mitchell StreetNorth WardTOWNSVILLE QLD 4810
Phone: 07 4722 5771 or 07 4781 4181Fax: 07 4722 [email protected]
for all skin or toe-clip samples:
Rick SpeareSchool of Public Healthand Tropical MedicineJCU House — Suite 1357 Mitchell StreetNorth WardTOWNSVILLE QLD 4810Phone: 07 4722 5777Fax: 07 4722 [email protected]
Michael MahonySchool of Biological SciencesUniversity of NewcastleCALLAGHAN NSW 2308
Phone: 02 4921 6014Fax: 02 4921 [email protected]
For reimbursement of costs associatedwith the transport of sick and dead frogscosts contact:
Nick SheppardThreatened Species UnitNSW National Parks and Wildlife Service(Northern Directorate)Locked Bag 914COFFS HARBOUR NSW 2450Phone: 02 6659 8231Fax: 02 6651 [email protected]
For information on frog keeping licencesand approvals to move some species ofdisplaced frog contact:
The ManagerNPWS Wildlife LicensingBiodiversity Research andManagement DivisionPO Box 1967Hurstville NSW 2220Ph 02 9585 6481Fax 02 9585 [email protected]
For information on the possible identity ofdisplaced frogs contact:
Frog and Tadpole Society (FATS)Frogwatch HelplinePh: 0419 249 728
designated sick and dead frog recipientsAlways contact therelevant specialistprior to sending asick or dead frog. Insome cases, onlywild frogs will beassessed for disease.Analysis may alsoattract a small feeper sample.
appendix 3
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The NSW Animal Welfare AdvisoryCouncil Procedure for humanelyeuthanasing cane toads or terminally illfrogs is stated as follows:• Using gloves, or some other implement,
place cane toad or terminally ill froginto a plastic bag.
• Cool in the refrigerator to 4°C.• Crush cranium with a swift blow using
a blunt instrument.
Note: Before killing any frog presumed tobe a cane toad, ensure that it has beencorrectly identified and if outside thenormal range for cane toads in NSW(north coast) that local NPWS regionaloffice is informed.
NSW Animal Welfare Advisory Council methodology
15
Northern NSW
ASR (Australian Seabird Rescue), ‘Waverly’,Pacific Highway, WEST BALLINA 2478
FRIENDS OF THE KOALA,PO Box 5034, EAST LISMORE 2477
KPSN (Koala Preservation Society of NSW),PO BOX 236, PORT MACQUARIE 2444
NRWC (Northern Rivers Wildlife Carers),PO Box 6432, LISMORE SOUTH 2480
NTWC (Northern Tablelands WildlifeCarers), PO Box 550, ARMIDALE 2350
TVWC (Tweed Valley Wildlife Carers),PO Box 898, MURWILLUMBAH 2484
WCGI (Wildlife Carers of Glen Innes),PO Box 520, GLEN INNES 2370
Central Coast including Sydney
AWARE (Aust. Wildlife Ambulance RescueService), PO Box 592, CARINGBAH 2229
AWH (Australian Wildlife Hospital),PO Box 84, RAYMOND TERRACE 2324
CCFFC (Cabramatta Creek Flying-foxCommittee), PO Box 430 BONNYRIGG 2177
FAWNA (For Aust Wildlife Needing Aid),PO Box 41, BEECHWOOD 2446
GLWR (Great Lakes Wildlife Rescue), c/-Huntley, The Lakes Way, BUNGWAHL 2423
HKPS (Hunter Koala Preservation Society),PO Box 544, RAYMOND TERRACE 2324
IFAW (International Fund for Animal WelfareAust P/L), 29 Georgina St, NEWTOWN 2042
KBCC (Ku-ring-gai Bat Colony Committee),45 Highfield Road, LINDFIELD 2070
KPC (Kangaroo Protection Co-operative),GPO Box 3719, SYDNEY 2001
NATF (Native Animal Trust Fund),8 Conway St, TORONTO 2283
ORRCA (Org. for the Rescue and Research ofCetaceans), PO Box 442, ARTARMON 2064
SMWS (Sydney Metropolitan WildlifeServices), 31 Chiltern Rd, INGLESIDE 2101
WILD & FREE,PO Box 268, GLOUCESTER 2422
WILDLIFE ARC (Wildlife - Animal Rescue &Care), PO Box 2383, GOSFORD 2250
WIRES (Wildlife Information & RescueService), PO Box 260, FORESTVILLE 2087(Note: WIRES has 25 Branches – addressesavailable separately)
Southern NSW
LAOKO (Looking After Our KosciuskoOrphans), 18 Kurrajong St, JINDABYNE 2627
NANA (Native Animal Network Assoc.),P.O. Box 780, ULLADULLA 2539
SWCG (Sunraysia Wildlife Carers Group),PO Box 189, GOL GOL 2738
Western NSW
LWC (Lachland Wildlife Carers),‘Revenue’, CONDOBOLIN 2877
WILDCARE QUEANBEYAN,PO Box 1404, QUEANBEYAN 2620
RRANA (Rescue & Rehabilitation ofAustralian Native Animals),107 Boughtman St, BROKEN HILL 2880
RSPCA (ACT) Inc.PO Box 3082WESTON ACT 2611
WCN(CW) (Wildlife Carers Network(Central West)), ‘Grunty Fen’,RUNNING STREAM, NSW 2850
appendix 4licensed wildlife carer and rescue organisationsFollowing is a list of wildlife rehabilitation groups licensed by
NSW National Parks and Wildlife Service4:
4 Note: some of these organisations may not care for frogs.
appendix 5 — sick or dead frog collection form
Sender details:
name: address: postcode:
phone: (w) (h) fax: email:
Collector details: (where different to sender)
name: address: postcode:
phone: (w) (h) fax: email:
Specimen details:
record no: no. of specimens: species name: date collected:day/month/year
time collected: sex: status at time of collection: date sent:male/female healthy(H)/ sick(S)/ dead(D) day/month/year
location: map grid reference:(easting) (northing)
reason for collection:
Batch details for multiple species collection:
species no. locality (AMG) date sex status (H/S/D)
habitat type: vegetation type: micro habitat:eg creek, swamp, forest eg rainforest, sedgeland eg creek bank, under log, amongst emergent vegetation,
on ground in the open
unusual behaviour of sick frogs:eg lethargic, convulsions, sitting in the open during the day, showing little or no movement when touched.
dead frogs appearance:eg thin, reddening of skin on belly and/or toes, red spots, sore, lumps or discolouration on skin
deformed frogs: dead/sick tadpoles:eg limb(s) missing, abnormal shape or length eg numbers/behaviour
unusual appearance of egg masses: recent use of agricultural chemicals in area:eg grey or white eggs eg pesticides, herbicides, fertilisers
other potential causes of sickness/mortality/comments/additional information:
NSWNATIONALPARKS ANDWILDLIFESERVICE
General inquiries: PO Box 1967 Hurstville NSW 2220Phone: 1300 36 1967
Fax: 02 9585 6555 Web site: www.npws.nsw.gov.au
© August 2001. Design and illustration by Site Specific Pty Ltd.Printed on recycled paper.