- 3.1 Physiology & Reproduction
- Gametes and embryos in the female reproductive tract.
- Blocking embryonic development in the brushtail possum by targeting the mucoid and shell coats, the extracellular matrix and LIF protein.
- Immune responses in pouch young and adult possums.
- Synchronised oestrus and controlled mating in possums and oral delivery of biocontrol agents.
- Immunoglobulin transfer in the mammary gland of the brushtail possum.
- Control of reproduction by targeting the possums pituitary gland through the use of GnRH-toxin conjugates.
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.
The potential reduction in returns from agricultural production, in the event that restrictions on access for meat and dairy products were imposed by our trading partners due to the presence of bovine Tb, could be 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 and 1999.
The NSSC is being transformed from a ministerial committee to a committee reporting to the Board of the Foundation for Research Science and Technology. The programme has received $2 million a year since July 1993 and has guaranteed funding only until the end of June 2000.
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.
Progress is monitored and research reports assessed by six reviewers. 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, Richard Sadleir and Hugh Davies 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 public informed about research in progress are being explored.
3.1 Physiology & Reproduction
3.1.1 PBC 201
Programme Title: |
Gametes and embryos in the female reproductive tract. |
| Programme Leader: | Professor John Rodger |
| Institution: | Marsupial CRC |
Programme Goal: Develop cost-effective in vivo and in vitro models of sperm and egg maturation, fertilization and embryogenesis in the possum to identify, test and develop promising reproduction-based biocontrols.
Objective 1
Objective Title: In vivo studies.
Research Leader: Dr Frank Molinia
Description:
An extensive suite of assisted reproductive technologies (ARTs) has been established to control possum reproduction in captivity and increase animal productivity all year round. These ARTs will be consolidated to develop in vivo models of a broad range of key reproductive events to pinpoint targets for possum biocontrol, and as tools for testing these targets and those identified by other biocontrol researchers.
Objective 2
Objective Title: In vitro studies.
Research Leader: Dr Kuldip Sidhu/Dr Karen Mate
Description
Development of in vitro model systems of key reproductive processes (egg and sperm maturation, fertilization and embryogenesis) will rapidly progress identification of suitable targets for possum biocontrol. Once established, these promise to be most efficient means for screening emerging vaccines.
3.1.2 PBC 203
Programme Goal: To clone genes, prepare and evaluate proteins that are essential for embryonic development:- Oocyte ECM (extracellular matrix), mucoid and shell egg coats, LIF (leukemia inhibitory factor).
Objective 1
Objective Title: ECM; Clone and characterise genes, prepare recombinant proteins.
Research Leader: Dr Lynne Selwood/Dr Shuliang Cui
Description:
To complete ECM amino acid sequence analysis, clone and characterise gene/s and prepare recombinant protein for antibody preparation. The previous project determined that the ECM is formed in the oocyte during oogenesis and secreted by the conceptus during early cleavage and that polarised secretion of ECM is essential for normal blastocyst development and possibly oogenesis. Principal research components would be: Completion of sequence analysis of ECM amino acids including additional bands obtained from the April 1999 field trip; use appropriate sequences for design of degenerate primers, clone genes using 3'RACE or library screening; characterise genes; prepare recombinant proteins; develop in vitro testing protocol.
Objective 2
Objective Title: Coat proteins: Complete cloning, characterise genes; prepare recombinant proteins; begin antibody production.
Research Leader: Dr Lynne Selwood/Dr Shuliang Cui
Description
Finish cloning of possum coat protein genes and prepare recombinant proteins of selected genes based on characterisation for antibody production. If mucoid and shell coats are to be used as targets in contraception, the effects would be two-fold; to inhibit production of the coats in the tract and to interfere with normal embryonic function as previously shown (Selwood, 1989, Gamete Res. 23; 399; Selwood et al, NSSC Workshop 1997). The research would consist of completion of cloning and characterisation to include both mucoid and shell coat proteins by 3'RACE or library screening, preparation of recombinant proteins; commence preparation of monoclonal antibodies and begin immune trials in vivo, adapt oviduct and uterine monolayer techniques to the possum for in vitro trials in 2000/1 when coat antibodies would be tested on coat production and embryonic development in vitro.
Objective 3
Objective Title: Preparation of LIF antibodies from LIF protein, assessment of effect in vitro and of time of expression of LIF in vivo in a staged series.
Research Leader: Dr Lynne Selwood/Dr Shuliang Cui
Description
Preparation and assessment of LIF monoclonal antibodies. LIF protein is a polyfunctional cytokine, which, in the mouse, suppresses differentiation of embryonic stem cells in vitro, inhibits proliferation of leukaemic cells and is essential for implantation. It plays a role in hypoblast formation and it is secreted by extra embryonic cells as well as by the uterus. Its function in marsupials in unknown but Dr. Cui et al (unpublished) have shown that it is a powerful suppressor of murine ES cell differentiation. The research would involve preparation of monoclonal antibodies using recombinant LIF protein, establishment of fibroblast feeder layers (FFL) to test the effect of LIF antibodies on LIF production, collection and assay of presence of LIF protein in a staged series of embryos and reproductive tract, immune trials of LIF proteins on possums in vivo.
3.1.3 PBC 204
Programme Title: |
Immune responses in pouch young and adult possums. |
| Programme Leader: | Professor Des Cooper |
| Institution: | Macquarie University |
Programme Goal: To study aspects of immune responses in both pouch young (PY) and adult possums which could be important for implementing biocontrol.
Objective 1
Objective Title: Study of aspects of adult possum immune responses.
Description:
A study of aspects of adult possum immune responses with emphasis on:
- the precise time at which PY become immunologically competent,
- the route of administration of antigens,
- individual variability in responsiveness,
- the association between major histocompatibility complex (MHC) type and immune response.
Objective 2
Objective Title: Immunoglobulin (Ig) mRNA and Ig protein.
Description
To define the stage at which (Ig) mRNA and Ig protein are first expressed in various lymphoid tissues.
Riboprobes and antisera specific for each class of Ig will be generated. PY from 1-200 days will be collected and stored as aliquots in liquid nitrogen until used. Reverse transcriptase polymerase chain reactions (RT PCR) will be used for qualitative analysis and Western blots will be carried out. Both techniques will also be used on adult tissue. Validation and optimisation of techniques will be first carried out on adult tissue before being applied to PY tissue which is very limited in supply.
3.1.4 PBC 205
Programme Title: |
Synchronised oestrus and controlled mating in possums and oral delivery of biocontrol agents. |
| Programme Leader: | Dr Bernie McLeod |
| Institution: | AgResearch, Invermay |
Programme Goal: To develop hormonal methods of controlling the time of oestrus, ovulation and fertile mating in captive possums and to establish formulation strategies to deliver effective doses of peptides and proteins across the possum gastrointestinal tract.
Objective 1
Objective Title: Controlling ovarian follicle populations.
Description:
A reliable method of controlling the time of ovulation in eutherian species is to block preovulatory follicle development by the administration of exogenous progesterone for a prolonged period. When the source of progesterone is removed, the subsequent development of preovulatory follicles, and therefore the time of ovulation, is synchronised between treated animals. This approach does not improve the synchrony of ovulation is possums, probably due to variation between animals in the numbers, size and/or physiological status of the small to medium-sized follicles present at the time of progesterone withdrawal. Variation between animals in the time of ovulation would be further increased by the extended period of preovulatory follicle development in this species. In this objective, the effectiveness of suppressing gonadotrophin for extended periods to control follicle emergence will be assessed, along with the rate and synchrony of preovulatory development following withdrawal of these treatments.
Objective 2
Objective Title: Induced ovulation of mature preovulatory follicles.
Description:
Following the standard methods of synchronising ovulations in possums (removal of pouch young; removal of progesterone implants), preovulatory follicle development occurs albeit poorly synchronised between animals, with ovulation occurring in 8-16 days. Nevertheless, a proportion of animals (40-75%) will have a mature preovulatory follicle present on Day 8. By selecting these animals (identified by laparoscopy) and inducing rupture of their preovulatory follicles by hormonal treatment, it would be possible to induce ovulation with a high degree of synchrony.
Objective 3
Objective Title: Permeation enhancers, metabolic inhibitors and peptide uptake.
Description:
The rate of uptake of a bioactive compound across the gut wall is influenced by its permeablility and by the concentration of the compound in the region of the mucosal tissue. Permeation enhancers can be used to disrupt the integrity of the epithelial layer, thereby making it more permeable to the compound being delivered. In addition, co-administration of metabolic inhibitors should sustain high local concentrations of the bioactive to promote absorption. In this objective, the efficacy of permeation enhancers and metabolic inhibitors will be assessed using the in situ possum gut model to directly determine rate of uptake of the bioactive.
Objective 4
Objective Title: Targeted delivery of peptides and proteins to the possum colon.
Description:
When presented in an unprotected form, the oral delivery of a peptide or protein invariably results in more than 99% of the bioactive being metabolised in the stomach and small intestine. A critical component of formulating peptide or proteins for delivery to possums is the protection against this degradation, with the compound being released it its active form in the large intestine. In this objective, methods of encapsulating model peptides and proteins will be assessed, including determining their resistance to degradation in acid and protease-containing media.
3.1.5 PBC 206
Programme Title: |
Immunoglobulin transfer in the mammary gland of the brushtail possum. |
| Programme Leader: | Drs Jerome Demmer and Frances M. Adamski |
| Institution: | AgResearch, Ruakura |
Programme Goal: To determine the classes and quantities of immunoglobulins in milk, and to characterise the mechanism required to transport them into the milk for development of methods for the biological control of possums. The aim of this work is to establish assays where the interaction between IgG and its transporter (FcRn) can be measured and manipulated. These assay systems will be used to develop the test biocontrol agents targeted against or through the IgG-transporter.
Objective 1
Objective Title: Characterisation of early immune transfer to the possum pouch young.
Description:
Previous research suggested that the IgG transport pathway is an ideal pathway for manipulation in a biocontrol strategy. Secretion of IgG into possum milk as early as day 20 of lactation and elevated expression of the IgG-transporter during early lactation, suggests that IgG is an important component of possum colostrum. To establish firmly the utility of the IgG-transport pathway the researchers need to demonstrate conclusively that IgG is a major component of colostrum formed at the beginning of lactation. In this objective we will determine the concentration of IgG and IgA in possum milk obtained closer to parturition (day (1-20).
Objective 2
Objective Title: Development of assay systems to analyse the interaction between IgG and its transporter.
Description:
The researchers will set up assay systems where the interaction between IgG and its transporter can be analysed. These will be used to screen for and evaluate biocontrol agents (molecules and chemical compounds) that have the potential to affect pouch young (PY) health or fertility. They could include inhibitors of IgG transport or biocontrol agents that are transferred into milk by these transporters targeted against the PY. Two strategies for developing biocontrol agents will include:
- Using current scientific knowledge to produce designer molecules such as toxin-Fc-fragment (part of the IgG molecule that is bound by the IgG-transporter) fusion proteins and inhibitory mutant Fc-fragments.
- Using screening methods such as HTS (High Throughput Screening), phage display and peptide libraries to find novel biocontrol agents.
3.1.6 PBC 209
Programme Title: |
Control of reproduction by targeting the possums pituitary gland through the use of GnRH-toxin conjugates. |
| Programme Leader: | Dr Doug Eckery |
| Institution: | AgResearch, Wallaceville |
Programme Goal: To control reproduction by targeting the possum pituitary gland through the use of GnRH-toxin conjugates.
Objective 1
Objective Title: Internalisation of the GnRH receptor.
Description:
The premise for the use of GnRH-toxins is that after GnRH is bound to its receptor the GnRH-receptor complex is internalised into the cell. It is this mechanism of internalisation that the researcher will try to exploit by conjugating toxins to GnRH, thereby using GnRH as a vehicle to get toxins into specific cells. The rate at which the receptor-ligand complex is internalised could have profound effects on the dose of GnRH-toxin needed and the type of bond used for conjugation. For instance, if internalisation is very slow, it is possible that by the time the complex is internalised factors in the circulation may have been able to break the bond between the GnRH and toxin, such that the toxin is not introduced into the cell. The aim of this objective is to determine the rate of internalisation of GnRH receptor in possum pituitary cells.
Objective 2
Objective Title: Toxin screening.
Description:
At present researchers are using pokeweed anti-viral protein (PAP) to prove the concept that this technology will be effective in controlling possum fertility. However, PAP is a relatively large protein molecule (~30kD) which may be difficult to deliver to possums via baits (e.g. across the gut wall). RNase is an alternative toxin the researchers will also test that is about half the size of PAP. In this objective the researchers will establish assays to screen other toxins (alone or conjugated to GnRH) for their ability to kill cells.
Objective 3
Objective Title: Effects of GnRH-toxins in vitro.
Description:
Once suitable conjugates are identified in objective 2, the researchers will test the ability of those conjugates to disrupt the function of pituitary cells in vitro.
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