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The Brushtail Possum in New Zealand

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UC Agriculture & Natural Resources Proceedings of the Vertebrate Pest Conference Title Fertility Control for Wildlife Management The Brushtail Possum in New Zealand Permalink https://escholarship.org/uc/item/75344338 Journal Proceedings of the Vertebrate Pest Conference, 22(22) ISSN 0507-6773 Authors Cowan, Phil Duckworth, Janine Cui, Xianlan et al. Publication Date 2006 DOI 10.5070/V422110092 eScholarship.org Powered by the California Digital Library University of California Fertility Control for Wildlife Management – The Brushtail Possum in New Zealand PhilCowan,JanineDuckworth,XianlanCui, and FrankMolinia NationalResearchCentreforPossumBiocontrolatLandcareResearch,NewZealand WernerLubitz and PetraWalcher Bird-C,Klosterneuburg-Kritzendorf,andDept.ofMedicinalChemistry,UniversityofVienna,Austria ABSTRACT :Fertilitycontrolvaccinesareunderdevelopmentforanumberofpestwildlifespecies,mostlybasedonwholezona pellucida(ZP)orindividualZPproteins.Suchvaccinesmustbeeffective,longlasting,cheapandreadilydeployed.Oneapproach todeploymentisoraldeliveryinbaits.ThisisonestrategybeingtakeninNewZealandforcontrolofbrushtailpossums,2-to4-kg marsupialsintroducedfromAustraliainthe1850s,andnowmajorpestsofbothconservationandagriculturalproduction.New Zealandhashighlyeffectiveaerialandgroundsystemsfordeliveryoftoxicbaitstopossumsthatcouldbeadaptedreadilytodeliver vaccinebaits.RecenttrialsincaptivitywherefemalepossumswereimmunisedwithrecombinantpossumZP3andZP2proteins demonstrated70-75%reductionsinfertilityinnaturalandassistedbreedingtrials.Immunisationwithpossum-specificepitopesof theZP2andZP3proteinshasalsoprovedeffectiveatreducingthenumbersoffertilisedeggsrecoveredfromimmunisedfemales. Forfielddeliveryofanoralvaccine,weareinvestigatingtheuseofbacterial“ghosts”.Thesearetheemptycellwallsofbacteria thathavebeenmodifiedtoexpresspossumZPproteinsintheircellwalls.Thepossum’simmunesystemrecognisesthebacterial ghost as foreign, and produces antibodies against them. At the same time, it is tricked into developing antibodies against the possumeggproteins,causingacontraceptiveeffect.Inarecentproofofconcepttrial,femalepossumsimmunisedwithapossum ZP2-bacterial ghost vaccinebynasal spray showeda significantreductionin fertility. Oraldelivery mayrequireprotectionof vaccineghostsfromdegradation.Wehavedevelopedaprotectivesystemandarecurrentlyrepeatingthistrialusingoraldeliveryof theZP2ghostvaccine.Ourfuturepriorityisincreasingthevaccineefficacyandlongevityaheadoflimitedfieldtrialsin2009. KEY WORDS :antifertilityagents,biologicalcontrol,immunocontraception,Marsupialia,NewZealand,possums,safety, Trichosurusvulpecula ,vertebratepestcontrol Proc.22nd Vertebr.PestConf. (R.M.TimmandJ.M.O’Brien,Eds.) PublishedatUniv.ofCalif.,Davis. 2006.Pp.102-105. INTRODUCTION Part of the strategy is focussed on fertility control, and There is a widening range of species for which specifically on development of an oral, bait-delivered fertility control vaccines are under development as immunocontraceptivevaccine(Cowan2000,Duckworth alternatives to lethal methods of management. Most of et al . 2006). This paper reviews progress to date in these vaccines are based on porcine zona pellucida or development of such a vaccine for possums based on species-specificzonapellucidaproteins(e.g.,Kirkpatrick zonapellucidaantigens,includingrecentresearchonthe et al . 1996, Hardy et al . 2002, Kitchener et al . 2002). potentialofbacterialghosttechnology(Eko etal .1999, While immunisation against zona pellucida proteins is Walcher et al . 2004) as a platform for oral vaccine knowntoinduceinfertilityinawiderangeofeutherian delivery. mammals,littleisknownoftheeffectsofsuchvaccines onmarsupialspeciesdespiteanurgentmanagementneed METHODS for such non-lethal control technologies for pest Following on from recent characterisation of the marsupials such as the common brushtail possum structure of the possum ZP proteins (Mate and (Trichosurus vulpecula ) (Cowan 2000). Possums were McCartney1998,McCartneyandMate1999,Mate etal . introducedtoNewZealandfromtheirnativeAustraliain 2003, Cui and Duckworth 2005), various trials were the1850sandarenowmajorpestsofbothconservation conducted to examine the effects of alloimmunisation andagriculturalproduction(Cowan2005).Theydamage with purified recombinant possum ZP antigens or nativeforestsbybrowsing,preyonnativebirds,andare epitopesontheimmuneresponsesandfertilityoftreated theprincipalwildlifehostofbovinetuberculosis.About femalepossums. US$45millionisspentannuallyonlethalcontrolofthis Thegeneraltestingprotocolusedwildadultpossums pest, mostly trapping and poisoning (Morgan and housed indoors in individual cages for a physical and Hickling2000,Cowan2005). immunological acclimatisation period of six to twelve Since its inception in the 1990s, the research weeks (Buddle et al . 1992). Ten days before the programme on biological control of possums has been experimentalperiod,femalepossumsweretransferred,in guided by a National Science Strategy overseen by a pairsmatchedbybodyweight,into4m ×4moutdoor committee of key end-user management agencies pens with shelter and sacks for nesting. Possums were (www.frst.govt.nz/About/possum.cfm). In the strategy, fed adlibitum freshfruitandcereal-basedpossumpellets, biocontrolisjustoneapproach,butseenasakeyonefor withwaterfreelyavailable. along-termsolutiontoNewZealand’spossumproblem. Femalepossumswereassignedatrandomtoexperi- 102 mental groups and immunised with Freund’s-based Initial assessments of non-target safety were carried vaccine containing either ZP antigen or PBS with outusinglaboratorymiceanddomesticchickens.These adjuvant only (Control Group) in weeks 0, 3, and 6. were immunised by injection with Freund’s-based Vaccines were prepared by mixing each recombinant vaccine containing either possum ZP antigen/epitope or protein with phosphate buffered saline (PBS) and then PBSwithadjuvantonly(ControlGroup),withboostersin emulsifying the mixture with an equal volume of IFAatweeks2,4,and6.Miceandchickenswerethen completeFreund’sadjuvantfortheprimaryimmunisation bredtoassessanyeffectsonreproductiveperformance. andwithIncompleteFreund’sAdjuvant(IFA)forbooster All projects were approved by the Animal Ethics immunisations. Control animals received the same Committee of Landcare Research, Lincoln and were adjuvantsemulsifiedwithplainPBS. performed in accordance with the 1987 Animals Fortrialsthatinvolvedassessmentsofbacterialghosts Protection (Codes of Ethical Conduct) Regulations of expressing possum ZP proteins, individually housed NewZealand. femalepossumswereimmunisedbyvariousroutes(sub- cutaneous,nasal,and/ororal)atvariousdoses3timesat2 RESULTS weekly intervals, with control females given plain Es- Varioustrialswereconductedusingantigensbasedon cherischiacoli ghostsonly.Thenasal/conjunctivalroute possumZP3,theN-orC-terminalregionsofpossumZP- ofimmunisationevokedthestrongestimmuneresponse, 2,andpossumZP1.AnepitopeofpossumZP3,homolo- and so it was used in a trial to assess the effects on gous to one shown previously to produce infertility in fertilityofimmunisationwithbacterialghostsexpressing mice (Millar et al . 1989), was also tested. With the ZP2 proteins in the periplasmic space. Various gastric exceptionofZP1,allantigenstestedproducedsignificant acidinhibitorswerealsotestedfortheirabilitytoimprove reductionsof60-87%(P<0.05)inthenumberofpouch immuneresponsetobacterialghostsgivenorally. youngorembryosproducedbytreatedfemalescompared Immune responses in all trials were assessed by with control females (Table 1). Immunisation with ZP standard techniques such as indirect Enzyme-Linked antigensappearedtohavetwoeffectsinSO/AItrials–a Immunosorbant Assay (ELISA) and Western blotting reductioninnumbersofovulationsandareductioninthe (Duckworth et al . 1999, Mate et al . 2003). The percentageofovulatedeggsthatwerefertilised(Table1). secondaryantibodyforELISA(suppliedbyDavidKay, BacterialghostsexpressingN-orC-terminalhalves Newcastle University) was raised in sheep against of possum ZP2 evoked immune responses in most possum serum immunoglobulin (Ig) and reacted with possums.Thepercentageoffertilisedeggsdeclinedby possumIgG,IgA,andIgM. 37%inpossumimmunisedwithZP2-Cbacterialghosts Fertility was assessed in two ways. In natural (Table2;P<0.05).Thisisthefirstdemonstrationofa breedingtrials,amaturemalewasintroducedtothepens mucosally-deliveredvaccineforpossumsthatproduceda and left with females for the duration of the breeding significantreductioninfertility.Doseresponseandroute season, with pouches of females checked regularly and of immunisation trials aiming to improve immune any pouch youngremoved sothatfemaleswouldcome responsestoorallydeliveredghostsidentifiedanoptimal backintooestrusrepeatedly(Duckworth etal .1999).In dose of ghosts, which when given orally with a gastric SO/AI trials, females were treated with hormones to acidinhibitor,producesasstronganimmuneresponseas inducesuperovulationandthenartificiallyinseminatedas a similar dose given nasally/conjunctivally. An experi- previously described (Molinia et al . 1998) except a ment is currently in progress to assess the effects on vaginalratherthanuterineroutewasused.Possumswere fertility of oral immunisation with bacterial ghosts killed, and the reproductive tract was removed and expressingZP2andZP3usingthisoptimalprotocol. ovaries separated for assessment of numbers of Totestfornon-targeteffects,miceandchickenshave ovulations sites and remaining unovulated follicles (>2 been immunised by injection with ZP3, ZP3-epitope, mm). Oviducts, uteri, and the left
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