- 3. Biological Control of Possums
3. Biological Control of Possums
The introduced Australian brushtail possum (Trichosurus vulpecula) is a very serious pest in New Zealand. It causes extensive damage to indigenous forests and acts as the major vector of bovine Tb to cattle and deer.
Forest ecosystems are being drastically altered by the selective attention of possums that can eliminate favoured browse-species from whole tracts of forest. Possums also prey on birds.
Any restrictions on access for meat and dairy products imposed by our trading partners due to the presence of bovine Tb could potentially reduce returns from agricultural production by up to $500 million annually.
Currently about $45 million is spent annually on poisoning and trapping possums. However, this level of expenditure is not sustainable and these methods of control are becoming less socially and politically acceptable. Around $15 million is spent annually on research into control of possums and bovine Tb.
Research into possum biocontrol as a long-term cost-effective solution to the possum problem was initiated in 1993. The expectation is, however, that control will be, for the foreseeable future, dependent on a number of technologies used in an integrated manner.
The research into biological control of possums, funded through Vote: Agriculture was initiated following a National Science Strategy Committee (NSSC) meeting, held in October 1992, at which clear research priorities for biological control were established. Priorities were reviewed at NSSC workshops in 1995, 1997, 1999 and 2001.
The NSSC has been transformed from the previous ministerial committee to a committee reporting to the Board of the Foundation for Research Science and Technology.
Most of the research programmes follow one of two directions for possum biocontrol. The first involves targeting physiological pathways, such as the possum reproductive system, while the second is looking for potential vectors. Vector organisms could, possibly, be genetically manipulated in order to enable them to spread disruptive genes throughout the possum population. These genes would be expressed as biochemicals that would interfere with possum reproduction or health.
Most of the physiological research is focusing on the reproductive system. This includes studies on gametes, fertilization, embryonic development and endocrine control of reproduction. Essentially, the search is on for possum-specific physiological pathways that could be targeted for disruption and at this stage much of the research is fundamental.
Surveys of parasites and pathogens of possums have been carried out in Australia and New Zealand. One species of nematode parasite, which was found in the highest prevalence in Australia, appears to be absent here. Work is continuing to see whether it would be beneficial to introduce the nematode that could simply add to the load of parasites in the possum population or be used as a vector of disruptive genes.
In New Zealand the distribution of parasites was very uneven with some possum populations being free of significant parasites. Consideration is being given to introducing the missing parasites to these populations.
The most pathogenic agents found in possums to date are viruses. One causes a syndrome known as wobbly possum disease (WPD) while another causes a range of symptoms including diarrhoea. The virus responsible for WPD has been characterised and appears to be related to Borna virus that infects a number of animal species including humans. The relationship between the viruses is thought to be distant but work aimed at clarification of the relationship is in progress. Neither of the possum viruses, noted above, has been shown to be important in wild possum populations but both decimate captive colonies.
An adeno virus that is possum-specific has also been identified and it is thought to have potential as a vector if it can be grown in the laboratory.
Very good progress has been made in developing an understanding of fundamental possum physiology and several pathways have been identified which could be targeted for disruption. The most promising area is female reproductive physiology and a number of novel approaches to disruption are currently being researched both in Australia and New Zealand.
Six reviewers monitor progress and assess research reports. Two members of the group, Drs Paul Livingstone (AHB) and Peter Kettle (MAF) are members of the NSSC. The four other members are Drs Ken McNatty, Hugh Davies, Henrik Moller, and Professor John Marbrook who are contracted by MAF to provide a technical overview of the research falling within their speciality fields.
MAF is well aware that the issue of the public acceptability of new technologies is of vital importance if biocontrol methods are to be used against possums. Ways of keeping the public informed about research in progress are being explored.
3.1 Physiology & Reproduction
3.1.1 PBC 251
Programme Title: |
Blocking embryonic development in brushtail possums (2002/03) |
Programme Leader: |
Professor Lynne Selwood |
Institution: |
The University of Melbourne |
Programme Goal: Three highly possum-specific protein targets, will be tested to determine their antigenicity and their effects on embryonic development in the possum. Alternative systems of protein expression will be examined.
Unlike other aspects of reproduction, possum embryonic development has many unique developmental features that provide suitable targets for controlling reproduction. Our previous projects have examined three of these.
- The polarised oocyte molecules are responsible for lineage allocation into pluriblast (embryonic) and trophoblast (placental) tissues (Frankenberg and Selwood, 1998, 2001). Of 10 vesicle-associated molecules identified (9 Vesicle Associated Proteins (VAPs) and hyaluronate), four are worth testing because of possum specificity and/or embryo lethality effects (VAP5, VAP7, VASA protein, and fetoprotein).
- The outer egg coat proteins (CPs) are essential for normal embryonic development (Selwood, 2000) and for maintenance of blastocyst epithelia in the possum (Casey and Selwood, 2002). We have isolated 7 shell coat proteins yielding 5 amino acid sequences. Three unique proteins (CP4, CP2 and CP5) are suitable for further studies. CP4 is tested here.
- Leukaemia inhibitory factor (LIF) is present in the possum at the time of implantation (Cui and Selwood, 2000) and is essential for implantation (Stewart et al, 1992). In each group, genes have been cloned and recombinant protein expressed and purified for testing in possums.
This project examines VAP5, CP4, and LIF, and further characterises VASA. We expect that these target molecules will be possum specific.
Objective 1
Objective Title: |
Assessment of Vesicle Associated Protein ,VAP5 |
Research Leader: |
Professor Lynne Selwood, Dr. Shuliang Cui |
Description:
Aims: The gene vap5 has been cloned and a recombinant protein expressed. It is a protein with extensive possum unique sequences and was originally isolated from vesicle-rich oocyte fractions. We will complete assessment of possums’ immune response to VAP5 recombinant protein and begin assessment of VAP 5’s effect on embryonic development and oogenesis in vivo and in vitro. Alternative protein expression protocols will be tested.
Outcomes: Because these proteins are unique to marsupials and have a significant role to play in lineage allocation in embryonic development, we expect responses to them to include inhibition of normal lineage allocation in possum embryos. Because these proteins appear very early in oogenesis in primordial follicles, or earlier, it is possible that an immune response to them would also target oogenesis. A paper on vap5 will be prepared by June 2003.
Objective 2
Objective Title: |
Assessment of Coat Protein 4 (CP4) |
Research Leader: |
Professor Lynne Selwood, Dr. Shuliang Cui |
Description
Aims: The gene encoding the novel coat protein (CP4) has already been cloned, and characterized and recombinant protein has been expressed. We will test CP4’s ability to raise an immune response in cycling possums, examine its effect on breeding and further examine the effect of antibodies raised against it on uterine tissues in vitro Alternative protein expression protocols to facilitate production will be tested.
Outcomes: Because the shell coat is marsupial specific, and we have demonstrated earlier that it is essential for normal blastocyst formation and for maintenance of blastocyst epithelia (Selwood, 2000, Casey and Selwood, 2002), we expect an immune response to the shell coat proteins to terminate embryonic development well before implantation stages. We expect to have published on CP4 expression by June 2003.
Objective 3
Objective Title: |
Assessment and cloning of implantation proteins |
Research Leader: |
Dr. Shuliang Cui , Professor Lynne Selwood |
Description
On the basis of what is known of LIF biology, interfering with normal LIF function either through LIF or its receptor, should affect normal embryonic development at implantation stages (flat embryo) and also primordial germ cell (PGC) proliferation between the primitive streak stages to early days postpartum. We have confirmed that these are likely targets in the possum.
Aims: Using recombinant possum LIF protein we will examine whether LIF induces an immune response in reproducing possums and begin assessment of LIF’s effect on embryonic development and gametogenesis. We know that LIF enhances proliferation of PGC in vitro, so we will test whether anti-LIF antibody can counter this effect. We aim to test alternative protein expression protocols to facilitate production.
Outcomes: As LIF is essential for implantation and PGC cell proliferation and the absence of its receptor has embryo-lethal effects, we expect the immune response to LIF or its receptor to cause embryonic development to fail and germ cell proliferation to be reduced. A paper on LIF function in vitro will be submitted by June 2003.
3.1.2 PBC 254
Programme Title: |
Control of reproduction in possums by targeting the oocyte-specific growth factors, GDF-9 and GDF-9B |
Programme Leader: |
Dr Doug Eckery |
Institution: |
AgResearch |
Programme Goal: |
To sterilise female possums by immunising them against GDF 9 and/or GDF-9B. |
Description:
Both GDF-9 and GDF-9B are oocyte-specific growth factors. Mutations in these growth factors or their absence can lead to infertility by blocking early follicular growth. The aim of this work is to determine the effects of immunising possums against these growth factors on ovarian function and reproductive activity.
Objective 1
Objective Title: |
Immunisation of possums against GDF-9 and GDF-9B |
Objective Leader: |
Dr. Doug Eckery |
Description:
Mutations in GDF-9 or GDF-9B or their absence can lead to infertility by blocking early follicular growth. In a previous study, we reported that immunisation of possums against a non-conserved region identified in each of these proteins showed no effects on reproductive activity; suggesting these regions were not important in the function of the proteins. The aim of this proposal is to determine the effects of immunising possums against the entire mature region of these growth factors on ovarian function.
Reporting Milestones
Date |
Description |
30 Sept 2002 |
Produce recombinant ovine GDF-9B. Immunise female possums against recombinant GDF-9B |
20 December 2002 |
Assess effects of immunisation on ovarian function |
31 March 2003 |
Produce recombinant ovine GDF-9. Immunise female possums against recombinant GDF-9. |
15 June 2003 |
Assess effects of immunisation on ovarian function Submission of final report. |
3.2 Microbiology/Parasitology
3.2.1 PBC 257
Programme Title: |
Transgenic worms for the control of possums. |
Programme Leader: |
Dr Warwick Grant |
Institution: |
AgResearch |
Programme Goal: To develop techniques that allow the production of transgenic Parastrongyloides trichosuri (a nematode parasite of possums) producing a protein(s) that interfere(s) with the growth, reproduction or longevity of its possum host.
Rationale: Sustainable biological control of possums via immunocontraception or a similar means requires an efficient, self-disseminating vector that is safe, specific to possums and will persist in the possum population. A strong candidate for the role of vector for an immunocontraceptive protein (or toxin etc) is Parastrongyloides trichosuri.
P. trichosuri is a nematode parasite of the possum. It is widely distributed in possums on the North Island of New Zealand but is endemic in only one location on the South Island, presumably as the result of a recent introduction. The dynamics of the introduction and spread of the parasite into a naïve population is being studied and the results one year after a deliberate introduction suggest that P. trichosuri will be a sustainable, self-disseminating vector for biological control. Other favourable features of P. trichosuri are:
- there is strong evidence that it is host specific;
- it does not elicit a strong protective immune response in its host, so the host remains infected for long periods and is susceptible to re-infection i.e. the infection is chronic;
- it does, however, elicit a host antibody response, so a protein produced by the parasite is likely to be exposed to the host immune system;
- it does not cause serious pathology or morbidity in the host; and
- it is capable of completing an apparently indefinite number of free-living generations outside the host. This acts to sustain and amplify its presence in the environment once released.
Objective 1
Objective Title |
Biolistic bombardment as an alternative transformation technology |
Objective Leader: |
Dr Warwick Grant |
Description:
Microinjection is technically demanding and requires adult female parasites, which have to be recovered from infected possums. Transformation of the free-living stages of the nematode is likely to be simpler, faster and cheaper but to date microinjection of the adult free-living worms has been unsuccessful. Experiments testing electroporation as an alternative to microinjection were unsuccessful. Biolistic particle bombardment is an attractive alternative to microinjection: it can be applied to large populations of either free-living or parasitic worms and has been shown to yield low copy number, genetically stable integrated transgenesis in C elegans.
Objective 2
Objective Title: |
DNA vectors for controlled expression |
Research Leader: |
Dr Warwick Grant |
Description:
The delivery of a protein to possums via expression in P. trichosuri requires that the protein be expressed and secreted by the parasitic stages of the nematode. This in turn requires (a) that expression of the transgene is controlled by a promoter with activity in parasitic stages and (b) the transgene encode a secretory signal peptide fused to the protein of interest.
Objective 3
Objective Title: |
Transgenic worms in possums in containment |
Objective Leader: |
Dr Warwick Grant |
Description:
If we are successful in isolating a promoter that gives high levels of Beta-galactosidase expression in free-living cultures and infective larvae, and which is active in parasitic adults from RT-PCR analysis of the P. trichosuri gene (see objective 2), we will test the expression of the reporter gene driven by this promoter following infection of possums with the transgenic line. Possums will be infected, housed and necropsied to recover parasites under appropriate containment and only after approval for these experiments has been granted by ERMA. Expression will be monitored by a combination of staining for enzyme activity and RT-PCR. Presence of the transgene in the worms (and their progeny) will be monitored by genomic DNA PCR.
Note that this objective is contingent upon obtaining a transgenic line with much improved levels of Beta-galactosidase expression. It is preferable also that this line be genetically stable and thus most likely derived from biolistic transformation rather than microinjection.
Reporting Milestones
Date |
Description |
20 Dec 2002 |
Testing of biolistic bombardment as an alternative transformation technology. |
30 April 2003 |
Isolation and testing of new promoters for expression of transgenes. |
31 May 2003 |
Testing of suitable transgenic line(s) in possums under containment. |
15 June 2003 |
Submission of a final describing these activities |
3.3 Viruses
3.3.1 PBC 252
Programme Title: |
Viruses for Biological Control |
Programme Leader: |
Dr Tao Zheng |
Institution: |
AgResearch |
Programme Goal: To isolate viruses from possums, and assess their usefulness for the biological control of possums
Description:
The goal of this programme is to isolate viruses from possums, and assess their usefulness for the biological control of possums.
- Determine the sensitivity of a nested polymerase chain reaction (PCR) for detection herpesvirus polymerase in faecal samples, using macropodid herpesvirus -1 as a positive control.
- Establish a RT-PCR assay for the detection of coronaviruses based on degenerated primers and determine its sensitivity in detection of coronaviruses in faecal samples, using a bovine coronavirus as a positive control.
- Detect herpesvirues and coronaviruses in possum faecal samples collected from different geographical areas by PCR and RT-PCR.
- Culture any PCR/RT-PCR positive possum faecal samples on cell lines for the isolation of herpesvirus/coronavirus.
- Determine current prevalence of anti-herpesvirus antibody in possum from Shannon by ELISA using antigens from a wallaby herpesvirus.
- Culture 50 possum faecal samples collected from areas where cross-reactive antibodies to antigens of a wallaby herpesvirus have been identified previously.
Reporting Milestones
Date |
Description |
30 Sept 2002 |
Determine the sensitivity of a nested PCR for the detection of herpesvirus in possum faecal samples. |
20 Dec 2002 |
Establish a RT-PCR assay for the detection of coronaviruses and determine its sensitivity in the detection of coronaviruses in possum faecal samples |
31 Mar 2003 |
Culture 50 faecal samples collected from areas where cross-reactive antibodies to antigens of a wallaby herpesvirus have been identified previously. |
15 June 2003 |
Submission of final report |
3.4 Vaccine Development
3.4.1 PBC 258
Programme Title: |
Improved diagnostic tests and vaccines for control of bovine tuberculosis. |
Programme Leader: |
Dr Bryce Buddle |
Institution: |
AgResearch |
Programme Goal: Develop and evaluate diagnostic tests and vaccines for control of bovine tuberculosis in cattle and possums.
Develop methods for measuring mucosal immune response in possums and delivery of vaccines for biocontrol of possums.
Objective 1
Objective Title: |
Immunology of bovine tuberculosis |
a) Improved diagnostic tests and vaccines for control of bovine tuberculosis
Description:
Develop and evaluate diagnostic tests and vaccines for control of bovine tuberculosis in cattle and possums.
- Establish whether the addition of dead BCG to an encapsulated oral bait vaccine lowers the effectiveness of the live BCG vaccine for possums and determine the optimal amount of live BCG in oral bait for protection against tuberculosis.
- Compare the efficacy of a newly derived attenuated M. bovis strain to BCG for protection of possums against bovine tuberculosis when the vaccines are administered as oral bait.
- Determine which possum gut associated lymphoid tissues are sensitised to M. bovis antigens following administration of oral bait BCG vaccine, with the aim of optimising the uptake of BCG in the gut where the delivery of BCG can be altered by varying the encapsulation process.
- Plan a field trial to determine the efficacy of an oral bait BCG for wild possums.
- Determine whether the addition of zinc ions to tuberculin increases the sensitivity of the tuberculin skin test response in M. bovis-infected animals.
- Compare the effectiveness of an ESAT-6/CFP10 interferon-g test with the standard interferon-g test for the differentiation of cattle infected with M. bovis and those exposed to environmental mycobacteria (part funded by AHB).
- Identify specific host genes from peripheral blood mononuclear cells (PBMCs) of M. bovis-infected cattle, which are up or down-regulated when re-exposed to M. bovis antigens. Analyse gene expression using DNA micro-arrays on total PBMCs.
- Complete the study to identify adjuvants and immunostimulants which induce strong cellular immune responses to M. bovis antigens in cattle and evaluate the best combinations of these in vaccines for protection against bovine tuberculosis (funding predominantly from DEFRA, UK).
Submit three papers to refereed journals.
b) Possum Immunology
Description:
Develop methods for measuring mucosal immune response in possums and identify vaccine delivery systems for biocontrol vaccines.
- Develop a serological test to measure immune responses of possums to a possum specific nematode which may be used as a vector for a biocontrol vaccine (collaboration with Mark Ralston).
- Determine the immune responses and hormone levels in possums following vaccination with a conjugated GnRH vaccine (collaboration with Ken McNatty and Doug Eckery).
- Test the effectiveness of including adjuvants and immunostimulants into a zona pellucida vaccine to enhance immune responses following oral delivery to possums (collaboration with Janine Duckworth and Phil Cowan).
- Submit one paper to a refereed journal.
Reporting Milestones
Date |
Description |
30 Sept 2002 |
Determine whether the addition of zinc ions to tuberculin increases the sensitivity of the tuberculin skin test response in M. bovis-infected animals |
20 Dec 2002 |
Determine the immune responses and hormone levels in possums following vaccination with a conjugated GnRH vaccine |
31 Mar 2003 |
Compare the efficacy of a newly derived attenuated M. bovis strain to BCG for protection of possums against bovine tuberculosis when the vaccines are administered as oral bait |
15 June 2003 |
Submit final report |
Objective 1
Objective Title: |
Immunology of bovine tuberculosis |
Research Leader: |
Dr Bryce Buddle |
Description:
Develop and evaluate diagnostic tests and vaccines for control of bovine tuberculosis in cattle and possums.
- Determine whether vaccination with a DNA vaccine and revaccination with BCG vaccine induces a greater level of protection in cattle against bovine tuberculosis than BCG alone.
- Determine whether age at which calves are vaccinated with BCG is a critical factor in protection against bovine tuberculosis, particularly when they have been sensitised to environmental mycobacteria.
- Determine whether the frequency of IFNg producing cells as detected by ELISpot correlates with protection against bovine tuberculosis in cattle.
- Cannulate the afferent lymphatic duct of the prescapular lymph node of calves to directly measure the immune responses to BCG vaccination.
- Determine whether a combination of ESAT-6 and CFP10 used in the interferong test can be useful in differentiating cattle infected with M. bovis from those infected with environmental mycobacteria.
- Establish whether vaccination of possums with a recombinant BCG expressing a possum cytokine, TNF-a, is more effective in protecting possums against bovine tuberculosis than BCG.
- Compare the efficacy of four different oral bait BCG vaccines prepared by Frank Aldwell (University of Otago) by challenging vaccinated possums with an aerosol of M. bovis.
- Determine the optimal dose of BCG in an encapsulated oral bait vaccine for protection against tuberculosis by vaccinating groups of possums with one of three doses of BCG and later challenging them with M. bovis.
- Submit three papers to refereed journals
Possum Immunology
Description:
Develop methods for measuring mucosal immune response in possums and use them to determine how responses are regulated by possum cytokines. Develop vaccine delivery systems suitable for combined delivery of tuberculosis and biocontrol vaccines.
- Produce one new recombinant possum cytokine and determine whether it enhances mucosal immune responses.
- Use immunohistochemistry to locate T cells in Peyer’s patches and mesenteric lymph nodes after oral immunisation with BCG.
- Determine the feasibility of using a recombinant BCG vaccine for protecting possums against tuberculosis and inducing an appropriate antibody response to achieve immunosterilisation of possums.
- Submit one paper to a refereed journal.
3.4.2 PBC 256
Programme Title: |
Oral Delivery of bioactives for Possums. |
Programme Leader: |
Dr Bernie McLeod |
Institution: |
AgResearch |
Programme Goal: To develop formulation strategies for oral delivery of biocontrol agents to possums, that maximise stability of the agent and promote its release and uptake in targeted regions of the gut.
Rationale
In the short to medium term, possum biocontrol agents will need to be delivered in bait and appropriate formulation techniques are required for this to be possible. Previously, we demonstrated that metabolism of peptides and proteins are many times lower in the hindgut (colon and caecum) than in the small intestine, identifying it as the appropriate region to target for oral delivery. The objective is to develop formulation strategies that will protect the bioactive during its passage through the stomach and small intestine and promote its release in the caecum and colon. The objective is to develop pelleted formulations, using selected polymers, that disintegrate and release bioactive in the hindgut. To date, we have produced formulations that offer protection in the stomach and small intestine, but further development is required to achieve suitable disintegration rates in the hindgut. To this end, additional polymers (chitosan, cellulose, pectin) and modification to formulation excipients (especially the type and amount of plasticiser) will be assessed. Degradation of the tableted formulations will be assessed both in vitro and in vivo.
The successful delivery of these formulations is dependent on understanding gastrointestinal transit in possums and how particle size influences this. We have investigated this for solutions and for a limited range of particles. Transit and residence times will now be determined for particulates ranging in size from 0.2mm to 2-3mm, using radio-labelled and radio-opaque markers.
Objective 1
Objective Title: |
Formulation for delivery to hindgut |
Objective Leader: |
Prof IG Tucker |
Description:
We have developed some matrix and polymer-coated pelleted formulations for possums. These have proved stable in acid and in luminal contents, but do not degrade sufficiently rapidly in lumen contents of the caecum and colon. New matrix and polymer-coated pellet formulations will be developed, using different polymers (chitosan, cellulose, and pectin) and these will be tested both in vitro and in vivo.
Objective 2
Objective Title: |
Targeting specific regions of gut |
Objective Leader: |
Dr Bernie McLeod |
Description:
To successfully design formulations that will target delivery to the possum hindgut requires detailed information of gastrointestinal transit of particles of different sizes. We have collected preliminary data for some particle sizes, but need to extend the size range and assess the influence of food intake. Transit of particles ranging from 0.2 to 2-3mm will be determined, monitoring particles labelled with radioisotopes by gamma scintigraphy, or radio-opaque particles by CT scan.
Reporting Milestones
Date |
Description |
30 Sept 2002* |
(Obj 1) Determine transit and disintegration of selected matrix and polymer-coated and matrix pellets in vitro and in vivo. |
20 Dec 2002* |
(Obj 1) Determine transit and disintegration of selected matrix and polymer-coated and matrix pellets in vitro and in vivo. |
31 Mar 2003 |
(Obj 2) Determine stability of a peptide-lipid construct in lumen contents from possum intestine, and its uptake across intestinal mucosa |
15 June 2003 |
(Obj 2) Establish transit and residence times of particulates in the possum gastrointestinal tract |
* As each formulation is produced (Milestone 1) its stability and degradation characteristics will be tested (Milestone 2). Therefore, both of these milestones will be answered for particular formulations in September and for others in December.
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Monitoring and Evaluation
MAF Policy
PO Box 2526
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Phone: +64 4 894 0623
Fax: +64 4 894 0741
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