Immunocontraception of Eastern Grey kangaroos (Macropus giganteus) with recombinant brushtail possum (Trichosurus vulpecula) ZP3 protein

Journal of Reproductive Immunology 79 (2009) 156–162

Immunocontraception of Eastern Grey kangaroos (Macropus giganteus) with recombinant brushtail possum (Trichosurus vulpecula) ZP3 protein Anne L. Kitchener, Amanda Harman, David J. Kay, Carmen A. McCartney ∗ , Karen E. Mate, John C. Rodger Cooperative Research Centre for Conservation and Management of Marsupials, School of Environmental and Life Sciences, University of Newcastle, Callaghan, NSW 2308, Australia Received 23 January 2008; received in revised form 14 October 2008; accepted 14 October 2008

Abstract This study examined the potential of a recombinant marsupial zona pellucida 3 protein as a contraceptive vaccine for the Eastern Grey kangaroo, a marsupial that is locally overabundant in several regions of eastern Australia. First, a pilot study using porcine zona pellucidae (PZP) demonstrated that ZP proteins, primarily the ZP3 component of PZP, are highly immunogenic in the grey kangaroo and produce a long-lasting humoral response to a single immunisation, as found in other marsupials. Immunisation with 300 ␮g of a non-glycosylated recombinant brushtail possum ZP3 (recBP-ZP3) protein in complete Freund’s adjuvant produced a similar, significant and sustained antibody response, and none of the immunised kangaroos (n = 7) produced offspring during the following breeding season compared with four out of the six control animals. An epitope analysis of the B-cell response to recBP-ZP3 using a brushtail possum ZP3 identified numerous B-cell epitope regions clustered around the N- and C-terminal regions of the protein. Two regions of interest for further fertility vaccine development based on their immunogenicity and fertility trials and functional studies in other species were found to be immunogenic. These results suggest that immunocontraception based on targeting the ZP3 protein within the zona pellucida may be an effective strategy for fertility reduction in Eastern Grey kangaroos. © 2009 Elsevier Ireland Ltd. All rights reserved. Keywords: Marsupial; Fertility; Immunocontraception; Kangaroo; Zona pellucida

1. Introduction Marsupials are of fundamental importance to the Australian environment; however, several macropod species have become overly abundant in some areas because of environmental changes since European settlement ∗ Corresponding author at: School of Environmental and Life Sciences – Biology, University of Newcastle, NSW 2308, Australia. Tel.: +61 2 49217883; fax: +61 2 49216899. E-mail address: [email protected] (C.A. McCartney).

(Newsome, 1975; Anonymous, 1997; Dawson et al., 2005). In New Zealand, the common brushtail possum is a major introduced pest (Cowan and Tyndale-Biscoe, 1997). Fertility control offers a humane, non-lethal and potentially sustainable solution to marsupial overpopulation (Oogjes, 1997). Immunologically mediated contraception has the potential for specificity to species and reproductive function (Delves et al., 2002). The most effective target for immunologically based fertility control thus far has been the zona pellucida (ZP), an extracellular matrix secreted around mammalian oocytes during the

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early stages of ovarian development (Wassarman, 1988). Whole porcine ZP (PZP) was the first immunologically based contraceptive effectively used for the control of wildlife (Kirkpatrick et al., 1992) and it continues to find practical application (reviewed by Kirkpatrick et al., 1996). The contraceptive effects of PZP immunisation have also been demonstrated in two marsupial species, the common brushtail possum and a macropod, the tammar wallaby (Duckworth et al., 1999; Kitchener et al., 2002). Although effective, a PZP-based vaccine is not appropriate for the management of free ranging marsupials. This demands a more species-specific vaccine that can be mass produced and remotely delivered to animals spread across large and inaccessible areas (Rodger, 2003; Mate and Hinds, 2003). The PZP is composed of at least three proteins (PZP2, PZP3 and PZP4) and may include a fourth protein (PZP1), according to the system proposed by the nomenclature committee at the National Center for Biotechnology Information (NCBI) and Conner et al. (2005). Based on work in the brushtail possum, the marsupial ZP3 protein shares several structural features with the eutherian protein. These conserved features include a short signal sequence, a conserved ZP domain, and a hydrophobic region at the C terminus just downstream from a putative furin cleavage site (McCartney and Mate, 1999). Despite these similarities, the overall amino acid sequence identity between brushtail possum ZP3 and eutherian ZP3 is only 45–46% (McCartney and Mate, 1999), which is promising for at least some degree of taxon specificity in a ZP3-based marsupial immunocontraceptive. Immunisation with recombinant brushtail possum ZP3 (recBP-ZP3) elicited a strong humoral immune response and significantly reduced the fertility of female possums from 53% to 17% (Duckworth et al., 1999). In this present study we report the findings of immunisation using recBP-ZP3 in Eastern Grey kangaroos, a macropod species targeted for fertility-based population management. Prior to initiating the recBP-ZP3 immunisation and fertility study, a small pilot experiment was undertaken (using PZP as the antigen), the goals being to: (1) develop appropriate methods of capturing, treating and monitoring immune responses and fertility in the large and difficult to handle Eastern Grey kangaroo, and (2) to demonstrate that the immune responses to PZP were similar to those seen in the earlier study using the ‘laboratory model macropod’, the tammar wallaby (Kitchener et al., 2002). In addition, to identify any highly immunogenic regions within brushtail possum ZP3 that could be potentially useful for further contraceptive antigen development, a B-cell epitope analysis

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of a possum ZP3 peptide library with the Eastern Grey kangaroo anti-PZP and anti-recBP-ZP3 sera was carried out. 2. Materials and methods 2.1. Animals Eastern Grey kangaroos at Tidbinbilla Nature Reserve (Australian Capital Territory) were darted using zolazepam hydrochloride (Zoletil [10 mg kg−1 ]; Virbac, Peakhurst, Australia). They were then brought into a 2 ha enclosure, microchipped, ear tagged, had their pouch young removed to activate breeding and were randomly assigned to treatment groups. The presence of pouch young was also checked each time the animals were caught and pouch young resulting from embryos in diapause removed and euthanased by intraperitoneal overdose of sodium pentobarbitone. Grey kangaroos at Tidbinbilla Nature Reserve have a breeding peak between December and April. Grazing was supplemented with lucerne hay and kangaroo pellets (Gordons Speciality Stock Feeds, Yanderra, Australia) and water (ad libitum). The research was approved by the Committee for Ethics in Animal Experimentation, University of Canberra (#CEAE98/3). 2.2. Antigen preparation Porcine zona pellucidae (PZP) were collected as described previously (Kitchener et al., 2002). The PZP were resuspended in citrate buffer at a concentration of 1 × 104 PZP mL−1 and heat-solubilised at 70 ◦ C for 4 h for use in immunoassays. The protein concentration of heat-solubilised PZP was determined using a DC Protein Assay Kit (BioRad, Hercules, CA, USA). Recombinant brushtail possum ZP3 (recBP-ZP3) was produced in an Escherichia coli expression system and purified as described previously (McCartney and Mate, 1999; Mate et al., 2003). The recBP-ZP3 was solubilised in a Tris buffer containing 6 M urea and the concentration determined using a DC Protein Assay Kit (BioRad). This protocol for the preparation and solubilisation of recBP-ZP3 has been used in all immunisation studies in marsupials thus far (common brushtail possum, Duckworth et al., 1999; koala, Kitchener et al., unpublished observations) with no evidence of clinically significant impacts of the 6 M urea. However, for work currently in progress on a buccally delivered recBP-ZP3 vaccine the urea concentration has been reduced to 3 M by ultrafiltration as a means of minimising any undetected side effects.

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2.3. PZP immunisation pilot experiment

2.7. Serum ELISAs

To confirm that the response in the Eastern Grey kangaroo to immunisation with PZP was similar to that in tammar wallabies (Kitchener et al., 2002), and to gain some idea of the longevity of the immune response in kangaroos, six female kangaroos were allocated to two groups. Individuals in the first group were immunised with 100 ␮g PZP in 0.5 mL citrate buffer (2 mM Na3 citrate, 2 mM Na2 EDTA, 0.14 M NaCl, 3 mM KCl, 10 mM Na2 HPO4 , 2 mM KH2 PO4 ) emulsified with an equal volume of complete Freund’s adjuvant (CFA; Sigma Chemical Co., St. Louis, MO, USA). The control group received CFA and citrate buffer only. Immunisations were delivered subcutaneously to two sites in the groin and intramuscularly to two sites in the rump (0.25 mL per site).

Serum titres of PZP and recBP-ZP3 antibodies were determined by ELISA. Polystyrene 96-well plates (Cliniplate EB; MTX Lab Systems, Vienna, VA, USA) were coated with 50 ␮L of heat-solubilised PZP or recBPZP3 diluted to 1 ␮g mL−1 in 0.1 M Na2 PO4 (pH 9.0) and incubated overnight at 4 ◦ C. Plates were washed between incubations with 0.05% (v/v) polyoxyethylene sorbitan monolaurate (Tween-20; Calbiochem, San Diego, CA, USA) in PBS (PBS-T) and sera and antibodies were diluted in 1% (w/v) bovine serum albumin (BSA, Sigma) in PBS-T. A blocking step of 3% (w/v) BSA in PBS-T for 3 h at 37 ◦ C was followed by overnight incubation with kangaroo sera at 4 ◦ C. Each sample of kangaroo serum was applied in triplicate wells, 100 ␮L/well, with 10-fold serial dilutions ranging from 100 to 10−10 . This was followed by room temperature (RT) incubations of polyclonal sheep antisera to wallaby Ig (1/2000) for 1 h and donkey anti-sheep IgG (1/3000) conjugated to horseradish peroxidase (Sigma) for 30 min. After washing in PBS-T, the plates were rinsed with phosphate citrate buffer. The substrate O-phenylenediamine dihydrochloride (Sigma) was diluted in phosphate citrate buffer containing sodium perborate, added to plates for 20 min in the dark, then read at 450 nm (BioRad 550 Microplate reader). The time series for each animal was carried out as one run and compared with dilutions of reference sera from four untreated female animals included in each run. A positive result was defined as having a mean optical density (OD) greater than the mean OD plus three standard deviations of the reference sera at the same dilution.

2.4. Recombinant ZP3 immunisation The following year, two groups, each consisting of eight female grey kangaroos, were vaccinated with either 300 ␮g of recombinant possum ZP3 in 0.5 mL or 0.5 mL saline alone (controls), emulsified in an equal volume of CFA administered as for the PZP. Boosters were administered 12 weeks after the initial injection and just prior to the breeding season at 28 weeks. Booster immunisations were with incomplete Freund’s adjuvant on the contralateral side to the previous injection. 2.5. Serum collection Pre-immune blood samples were collected before initial immunisations and at approximately monthly intervals, weather permitting, except during the winter months. Blood samples (10 mL) were collected using a 21G needle via the jugular vein while the animals were anaesthetised. Blood was allowed to clot for a minimum of 30 min at 4 ◦ C before being spun at 700 × g. The serum was collected and stored at −20 ◦ C. 2.6. Breeding trial During the winter of the recBP-ZP3 trial, one treated animal and two control animals died, leaving seven recBP-ZP3 and six control animals, respectively for a breeding trial. After the 28-week booster injection two males were housed with the females (September 2000 to April 2001) and pouch checks made (under anaesthetic) to assess fertility.

2.8. Two-dimensional electrophoresis and Western blotting 2D polyacrylamide gel electrophoresis (PAGE) was carried out using heat-solubilised PZP that had been dialysed against Milli-Q water for 48 h at 4 ◦ C to remove salts before being freeze-dried overnight. Samples were rehydrated to a concentration of 160 ␮g mL−1 in buffer containing 8 M urea (Sigma), 2% (w/v) 3-[(3-cholamidopropyl) dimethylammonio]-1propansulfonate (CHAPS, Sigma), 10 mM dithiothreitol (DTT, Sigma), 2% (v/v) ampholytes (Biolyte 3/10, BioRad) and a trace of bromophenol blue. A total of 20 ␮g of rehydrated sample was used for isoelectric focussing followed by SDS-PAGE, as described previously (Kitchener et al., 2002). Gels were then either silver stained as per instructions (Amersham Pharmacia Silver staining kit) or blotted onto nitrocellulose

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membrane (BioRad) at 100 V for 1 h followed by 30 V overnight at 4 ◦ C in Tris glycine buffer containing 20% (v/v) methanol. Membranes were then washed in 0.05% (v/v) Tween in Tris buffered saline (TBS-T, 0.14 M NaCl, 3 mM KCl, 25 mM Tris [Sigma]), which was used for washing between incubations, markers were removed and stained with 0.1% amido black, and the remainder of the membrane was blocked in 5% skim milk powder in TBS-T for 2 h at RT. Membranes were incubated in kangaroo sera, from a sample time at which PZP-immunised kangaroos had high titres, diluted to 1/500 in 1% BSA in TBS-T at RT for 1 h. Similarly, the membranes were then incubated in sheep anti-wallaby Ig (1/400) and donkey anti-sheep IgG conjugated to alkaline phosphatase (1/15,000, Sigma). Western Blue Substrate (Promega, Madison, WI, USA) was added for 5 min before rinsing in water. 2.9. Epitope analysis A set of overlapping peptides (PepSet) designed to cover all possible epitopes was made by dividing the 422 amino acids of brushtail possum ZP3 into 83 dodecapeptides, offset by five amino acids. The brushtail possum ZP3 PepSet was synthesised in a cleaved format (Mimotopes Pty. Ltd., Clayton, Victoria, Australia) and each peptide was biotinylated at the N-terminal to facilitate binding to 96-well plates. Each lyophilised peptide was reconstituted in 200 ␮L of pure dimethyl sulphoxide (Sigma) and stored at −80 ◦ C. The final working solution of peptide was a 1/1000 dilution of the stock diluted in PBS containing 0.1% (v/v) Tween-20 and 0.1% sodium azide. Streptavidin-coated plates were prepared by adding 100 ␮L of 5 ␮g mL−1 streptavidin to each well of Nunc Immuno-Plate MaxiSorb F96 flat-bottomed plates (Medos, Mt. Waverly, Australia). The plates were left exposed to air at 37 ◦ C overnight to allow the solution to evaporate to dryness. Non-specific adsorption was blocked with PBS containing 1.0% (w/v) sodium caseinate, 0.1% (v/v) Tween-20 and 0.1% sodium azide shaken over night at 4 ◦ C. After washing, 100 ␮L of each peptide solution was added for 1 h at RT while shaking at 75 rpm. Plates were washed five times and dried at 37 ◦ C for 2 h and stored with silica gel at 4 ◦ C for up to 2 weeks. Antisera from control and immunised kangaroos were diluted 1/500 in PBS containing 0.1% (w/v) sodium caseinate, 0.1% (v/v) Tween-20 and 0.1% sodium azide and added overnight at 4 ◦ C, shaking at 75 rpm. Individual recBP-ZP3 antisera samples (n = 6) and pooled (n = 3) PZP antisera were tested in triplicate with the

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entire PepSet. After washing, sheep anti-wallaby Ig diluted at 1/1000 was added and incubated at RT for 1 h. The plates were washed six times and horseradish peroxidase labelled donkey anti-sheep IgG diluted to 1/2000 (Sigma), then added at RT for approximately 40 min. The remainder of the procedure was as described above for the serum titre ELISAs. As a background control the entire ELISA process was also applied to two wells that did not contain ZP3 peptides. Relative antibody binding levels (RAbB) were calculated by dividing the mean post-immune OD by the mean pre-immune OD and subtracting one. Mean RAbB and standard errors were calculated and plotted against peptide number using individual animal data (i.e., not from pooled sera). When using extensive PepSets, such as for possum ZP3, it is feasible to treat some of the peptides within the set as negative controls for the whole set (Geysen et al., 1987). This was done by taking the mean of the lowest quarter of RAbB values determined for the pooled samples plus three times the standard deviation of the mean of these values. This value represents the lower limit of RAbB values that was used to determine a positive response to a linear epitope. 3. Results 3.1. PZP pilot experiment Animals received an initial immunisation with PZP in Freund’s complete adjuvant and were monitored for the production of antibody to heat-solubilised PZP. Over the first month there was an increase in antibody titres to 10−8 , which dropped slightly to a plateau of 10−7 , where it remained stable for the remainder of the trial (13 months; data not shown). As antibody levels did not drop significantly, no boosters were given to these animals. The titres of control animals remained less than 10−1 throughout the trial (data not shown). Western blotting of 2D-electrophoresed PZP indicated that antibodies had been primarily made to porcine ZP3 (data not shown). 3.2. Eastern Grey kangaroos produced antibodies to recBP-ZP3 The serum antibody levels against recBP-ZP3 for both treated and control animals are shown in Fig. 1. The titres in treated animals were significantly higher than those of the controls (p < 0.01, n = 8, analysis of variance) 1 month after the initial immunisation. The pattern of the immune response found in previous work with macropods was repeated here. There was a sharp increase in the

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A.L. Kitchener et al. / Journal of Reproductive Immunology 79 (2009) 156–162 Table 1 The effect of immunisation with recombinant brushtail possum zona pellucida 3 protein (recBP-ZP3) on the production of pouch young by Eastern Grey kangaroos. Treatmenta

Control recBP-ZP3

Number of animals with pouch young at Time 0b

1 monthb

6 months

13 months

2/8 6/8

4/8 3/8

0/6 0/7

4/6 0/7c

a Initial immunisation at time 0; booster immunisations given at 3 and 6 months. b Any pouch young present were removed. c p < 0.05 (Chi-squared test with Yate’s correction).

Fig. 1. Time series of serum Ig antibody titres to recombinant brushtail possum zona pellucida 3 protein (recBP-ZP3) of Eastern Grey kangaroos immunised with recBP-ZP3 (–䊉–) or saline control (––). Each point consists of mean ± S.E.M. All titres, except for preimmune sera at time 0, were significantly higher in PZP-immunised kangaroos as a group than in saline-immunised animals, as tested by two-factor analysis of variance (treatment/time) with post hoc Student–Newman–Keuls. Initial immunisation took place on day 0 and boosters were administered at weeks 12 and 28.

levels of antibody produced 1 month after immunisation, which dropped slightly over the next month, with little response to subsequent booster immunisations, reaching a plateau at around a titre of 10−6 , which was maintained throughout the trial. 3.3. Immunisation with recBP-ZP3 reduces the fertility of Eastern Grey kangaroos No recBP-ZP3-immunised animals produced pouch young during the 2000/2001 breeding season, while four of the six control animals produced young (Table 1). This was statistically significant (p < 0.05) (Chi-squared test with Yate’s correction).

3.4. Epitope analysis of possum ZP3 using Eastern Grey kangaroo antisera Possum ZP3 epitope data from individual antisera collected from six kangaroos immunised with recBPZP3 are shown in Fig. 2. No significant binding to the possum ZP3 peptides was seen using sera from control animals. The mean RAbB to the ZP3 PepSet showed reactivity over several peptides (2–33) at the N-terminal and in more distinct regions, peptides 50–53, 62–63, 65, 70–72 and 80, at the C-terminal (Fig. 2a). There was a high degree of variability in RAbB to different peptides among individuals (Fig. 2a and b). Although reactivity is concentrated around the N-terminal and C-terminal regions, not all of the antisera react to the same peptides. Several (12) N-terminal peptides reacted with all six antisera; however, at the C-terminal only peptides 65 and 71 reacted with all antisera tested (Fig. 2b). Although limited RAbB occurred above the assigned lower limit in some samples, there was no significant binding to any of the brushtail possum ZP3 PepSet peptides using serum pooled from three kangaroos immunised with PZP (data not shown).

Fig. 2. A consensus graph of the data from antisera of individual Eastern Grey kangaroos immunised with recBP-ZP3. (a) The mean RAbB and standard error of six (6) Eastern Grey kangaroos plotted against PepSet peptide shows reactivity in a pattern similar to the pooled antisera, over several peptides (2–33) at the N-terminal and at peptides 50–53, 62–63, 65, 70–72 and 80 at the C-terminal. The x-axis crosses the y-axis at the lower limit RAbB of 0.818. (b) Those of the six (6) sera that have reacted above 0.818 RAbB plotted against each PepSet peptide. This graph shows that all peptides have reacted to at least one antiserum and some have reacted to all the antisera examined (e.g. peptides 2, 7 and 8).

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4. Discussion Immunisation with PZP or recBP-ZP3 produced a strong specific antibody response in Eastern Grey kangaroos in this study. In the case of recBP-ZP3, immunisation completely prevented production of pouch young for the duration of the trial. The serological response to PZP was strong in Eastern Grey kangaroos after the initial immunisation, similar to that observed in eutherian mammals (Sacco et al., 1981; Miller et al., 2000). However, unlike the classical response in eutherian mammals, the level of antibody production was maintained without diminution for at least 13 months. The progress of the immune response in Eastern Grey kangaroos appears to be very similar to the response to PZP in tammar wallabies (Kitchener et al., 2002) and brushtail possums (Duckworth et al., 1999). Peak titres are reached within 1 month of the initial immunisation followed by a slight fall to a plateau (approximately 10−7 ), which is maintained for several months. There is usually no significant effect of a booster, and indeed similar levels of antibody against PZP (around 10−7 ) were maintained in grey kangaroos in this study after a single immunisation. This lack of a clearly defined primary and secondary response to ZP proteins in marsupials remains consistent over several studies, and supports the belief that the marsupial immune response differs from eutherians, but is not necessarily weaker or slower, as previously suggested (Rowlands, 1970; Jansen et al., 1991). The sera from PZP immunised Eastern Grey kangaroos in this study only reacted to the PZP3 charge species in Western blotting. Serum from tammar wallabies immunised with PZP reacted with charge species of three glycoprotein families (PZP2, PZP3 and PZP4), although reactivity to PZP3 was the strongest (Kitchener et al., 2002). This is consistent with studies in eutherian mammals that have found ZP3 to be the most immunogenic of the three glycoproteins (Koyama et al., 1996). Despite the strong and predominant response to the PZP3 protein, there was no significant binding to any of the possum ZP3 PepSet with sera pooled from three PZPimmunised grey kangaroos. Unfortunately, at the time of the study the full amino acid sequence for Eastern Grey kangaroo ZP3 was not available to develop a grey kangaroo ZP3 PepSet, and thus grey kangaroo ZP3 B-cell epitopes recognised by anti-PZP serum may have gone undetected. However, immunisation of Eastern Grey kangaroos with a non-glycosylated possum ZP3 protein derived from a bacterial expression system induced a specific anti-ZP3 response of a similar magnitude to PZP, and

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produced a 100% reduction in fertility. The antibody response to recBP-ZP3 was maintained at high levels (>10−6 ) for the duration of the trial, 7 months after the final immunisation. The similarity in the immunogenicity and relative longevity of the response to both highly immunogenic glycosylated PZP and possum recombinant ZP3 is encouraging for future immunocontraceptive development. The search for a ZP-based immunocontraceptive that induces high levels of antibodies associated with infertility has resulted in an extensive B-cell epitope analysis of ZP3 from numerous eutherian species, e.g. mouse (Sun et al., 1999), pig (Afzalpurkar and Gupta, 1997), marmoset (Paterson et al., 1999) and humans (Paterson et al., 1998). This study has investigated marsupial ZP3 Bcell epitopes using polyclonal sera from ZP3-immunised kangaroos in order to identify specific immunogenic regions of the protein that may be involved in the reduction of fertility. Although a general pattern of reactivity was observed over several B-cell epitope regions, there was a strong reaction to two regions of interest, peptide 7 and peptides 71–72. Peptide 7, which begins eight amino acids downstream of the predicted signal sequence cleavage site of brushtail possum ZP3, is in a region (23 amino acids following the signal peptide) that is highly divergent among eutherian species (Swanson et al., 2001) and thought to be involved in human (Eidne et al., 2000) and porcine (Gupta et al., 1995) sperm–egg interaction. The functional role of this region is not known in marsupials, but limited amino acid identity at this site between marsupial families is suspected based on PCR analysis of ZP3 from several marsupial species (McCartney et al., 2007). There was also strong reactivity to another region of interest at peptides 71–72 incorporating some of the amino acids that make up a ZP3 peptide that has been tested in the brushtail possum. Immunisation with this peptide conjugated to keyhole limpet haemocyanin reduced the number of embryos produced by brushtail possums by 60% (Duckworth et al., 2001). This study has demonstrated the effectiveness of a non-glycosylated, bacterially produced, recombinant ZP3 protein as a potent and potentially long-lasting contraceptive in the Eastern Grey kangaroo. Studies to identify the critical epitopes of the ZP3 protein and adjuvants that are effective in macropod marsupials via mucosal routes are currently in progress. Acknowledgements This work was funded by the Australian Government’s Cooperative Research Centre (CRC) Program through the CRC for Conservation and Management of

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