5. Possum Biocontrol

5.1 PBC 261

Programme Title: Gametes and embryos in the female reproductive tract
Programme Leader: Professor John Rodger
Institution: Marsupial CRC Limited

Summary

This project is part of an integrated programme of research, towards development of fertility-based biocontrol for the possum. Apart from its role in marsupial conservation as an assisted breeding strategy, in vitro fertilisation (IVF) will be an important tool to identify suitable gamete antigen targets for brushtail possum biocontrol in New Zealand.

Background

The goal of the project is to develop cost-effective in vivo and in vitro models of sperm and egg maturation, fertilisation and embyrogenesis in the possum, to identify, test and develop promising reproduction-based biocontrols.

Approach & Outcomes

Objective 1

  • Towards a fully defined "off the shelf" IVF media, possum synthetic oviduct fluid was modified this year (pSOF II), based on most recent measures of ionic and energy substrate composition of possum oviduct fluid collected in vivo.
  • Cloning and sequencing of the possum LH receptor was completed this year to develop probes for in situ hybridisation studies of ovaries collected from superovulated possums. These will be used to determine the optimum time of ovarian LH expression, so that current superovulation treatments may be optimised to produce the maximum yield of matured eggs.
  • Collection of in vivo matured eggs from superovulated females, and in vivo capacitated sperm from the oviducts of artificially inseminated possums was used as the positive control for IVF experiments. This positive control is now established and no longer necessary for any future IVF trials, since in vivo sperm treatments result in consistent fertilisation rates.
  • A trial is underway to establish an in vivo model of embryogenesis. After removal of pouch young (current best method to synchronise oestrus), females will be artificially inseminated into the vagina at times that correspond with presumptive oestrus, to yield viable new pouch young.

Objective 2

  • Objective measures of sperm motility and viability of in vitro capacitated sperm derived from pSOF and EMEM based media were completed. Typical eutherian capacitation profiles (i.e.: changes from straight-line to circular motion followed by star-shaped hyperactivity), were not observed for the possum. However, on-going Marsupial CRC-funded work revealed that unusually low calcium levels are critical in the capacitation process.
  • Oviduct specific glycoprotein (OGP), has been identified in the possum as a promising oviduct target for biocontrol. Large-gel SDS page has been used to purify possum OGP, and these bands are currently being sequenced. A rapid dot-blot assay has been developed to estimate OGP, which, in conjunction with measures of calcium, will be used in the future as quality control measures for IVF media.
  • A comparison of EMEM with newly formulated pSOG II in IVF experiments, revealed that both media supported similar rates of sperm-egg binding, penetration and fusion. "Conditioning" of either media by the presence of oviduct epithelial cells, resulted in a change in the ionic composition that was directly related to IVF success. IVF experiments using in vitro matured eggs (collected from PMSG-primed females and cultured for approx. 40hr) demonstrated for the first time, fertility of possum eggs matured in culture.
  • A comparison of in vitro with in vivo fertilised eggs was completed. Under current IVF conditions, penetration of sperm is via a large hole of the zona pellucida and is usually polyspermic. Suprisingly, acrosome-intact sperm heads were found within the cytoplasm of these eggs, which raises major questions (and thus potential new targets) about the mechanism and site of sperm-egg membrane fusion in the possum.
  • A pilot study on embryo in vitro culture was completed. Recently fertilised eggs collected from the upper oviduct of artificially inseminated possums, developed to the 4-cell stage after culture on oviduct epithelial cell monolayers. The developmental capacity of later staged embryos in vitro will be assessed in the future.

5.2 PBC 263

Programme Title: Blocking embryonic development in Brushtail Possums
Programme Leader: Professor Lynne Selwood
Institution: University of Melbourne

Summary

Each of the targeted proteins has sufficient possum specificity and developmental significance to make them suitable for testing to determine their effect on possum fertility. Expression and other studies have indicated that their time of action will effectively cover the pre-implantation period and germ cell proliferation.

Background

The goal of this project was to characterise cloned genes, prepare and evaluate proteins that are essential for embryonic development by in vitro assay systems and examination of gene expression and protein secretion; vesicle-associated molecules (VAMs) in oocytes, mucoid and shell coats (CPs); and leukaemia inhibitory factor (LIF) are targeted.

To identify proteins that are essential for embryonic development and use these for immunocontraception of the brushtail possum. In our previous projects we established that VAMs, CPs and LIF, many of which are unique to marsupials, are essential for different stages of embryonic development in the brushtail possum. VAMs are essential for early embryonic development during cleavage and blastocyst stages, CPs are required for blastocyst formation and maintenance and LIF plays a role in suppression of differentiation and proliferation of germ cells and is essential for implantation.

Approach & Outcomes

We used differential polyacrylamide gel electrophoresis (PAGE) and amino acid sequencing to identify the possum proteins. Gene cloning and expression studies to identify their genes and confirm their presence in possums, protein expression systems to provide recombinant proteins for production of antibodies to test the effects of immunocontraceptive molecules in vitro. In vitro protocols for follicles, embryos, uterine and various stem cell-like types have been established for genes and proteins of interest.

We have identified 8 vesicle-associated molecules (VAMs), 7vesicle associated proteins (VAPs) and hyaluronate an ECM molecule and consider four of these are worth further testing because of possum specificity and/or embryo lethality effects (Vap5, Vap7, vasa protein, and alpha fetoprotein). VAP5 is a unique protein, its gene has been cloned, and the novel VAP5 recombinant protein produced. Its high antigenicity suggests VAP5 is a suitable target for intervening in normal development of oocytes and during cleavage in the possum. Other molecules which could be suitable are hyaluronate linker protein and inter trypsin inhibitor.

We have established for the possum that the shell coat is produced throughout the pre-implantation period with a 10x increase in volume at the trilaminar blastocyst stage before implantation and is essential for normal development. PAGE analysis followed by amino acid sequencing of CPs identified 5 proteins of which the fourth (cp4) has been cloned, and the recombinant protein and antibody produced. The first half of the molecule shows high antigenic patches while the second half shows high antigenicity throughout making the molecule suitable for immunocontraception. Work is in progress on other cloned fragments of CP2 and 5.

We have previously reported tvLIF cloning and characterisation. A preliminary study of the binding capacity of the monoclonal antibody detected a 65 kDa native LIF protein in uterine tissue. The tvLIF gene is expressed in uterine tissue constantly at a basal level. Semi-quantitative studies show that tvLIF is expressed in the uterus between the early unilaminar blastocyst and the late flat embryo stage. It is interesting that LIF expression is strongest at implantation even though implantation in the possum is extremely superficial. Possum LIF production responds to regulatory molecules in vitro. Possum LIF protein is necessary for proliferation of germ cells in vitro. These results suggest that LIF immunocontraception in addition to affecting implantation will affect the number germ cells produced in the gonads of both male and females.

Publications

Cui, S. and Selwood, L. (2000): cDNA cloning, characterisation, expression and recombinant protein production of leukemia inhibitory factor (LIF) from the marsupial, the brushtail possum, Trichosurus vulpecula. Gene, 243, 167-178

Fletcher, T.P. and Selwood, L. (2000): Possum Reproduction and Development. In "The Brushtail Possum in New Zealand". (Ed T. Montague), pp. 62-80. Landcare Press, New Zealand.

Selwood, L. (2000): Marsupial egg and embryo coats. Cells, Tissues, Organs. 166, 208-219

Casey, N. (2000): The role of the outer egg coats during pre-implantation development in the brushtail possum. MSc Thesis. La Trobe University 2000.

Frankenberg, S. and Selwood, L. (2001): Ultrastructure of oogenesis in the brushtail possum. Molecular Reproduction and Development. 58, 297-306.

Frankenberg, S., Tisdall, D. and Selwood, L. (2001): Identification of a homologue of POU5F1 (OCT3/4) in a marsupial the brushtail possum. Molecular Reproduction and Development.58, 255-261

5.3 PBC 264

Programme Title: Immune responses in pouch young and adult possums
Programme Leader: Des Cooper
Institution: Macquarie University

Summary

The overall goal of studying aspects of immune response of pouch young (PY) and adult possums, which could be important for implementing biocontrol, has been achieved. The first objective was to study aspects of the adult immune response with particular regard to the route of administration, individual variability in response and the role of the major histocompatibility complex (MHC). We elected NOT to use Freund's adjuvant in making our immunisations, in order to be as close as possible to the conditions under which immunocontraceptives would be used in the field. The antigen was MPT64, a myobacterial protein. We have been unable to get any humoral response by either DNA plasmid immunisation or via the mucosal route, which are routes under consideration for immunocontraception. The antigen we used, MPT64 evokes a strong humoral response in sheep when Mycobacterium bovis is injected as part of Freund's adjuvant. This occurred in the sheep antisera we made to IgG, IgA, IgM and IgE in the initial stage of the project. The anti-MPT 64 in the antisera gave us high control values, which delayed the project for 6-8 weeks until we discovered the problem.

As our work progressed evidence accumulated in the literature, which showed that in five species of mammal, including possums, even with Freund's, the level of female sterility obtained was not sufficient for long-term possum population reduction. We would probably have not undertaken the work Objective 1 had this information been available to us.

The second objective was to study expression of the genes for the four classes of heavy chains of possum immunoglobulin. Unambiguous results were obtained. IgM and IgA genes are switched on very early, but IgG and IgE genes are not expressed until the second half of the pouch life. Since the antibodies, which the mother transfers into the circulation of the pouch young via her milk are IgG (Adamski et al), this leads to a suggestion for a possible method of biocontrol: if a small molecular weight inhibitor of transfer of maternal IgG into the milk could be found, it could be used to compromise the pouch young through infectious disease. Previous work from this laboratory has shown that a large range of bacterial species, some potentially pathogenic occurs in the pouch.

Background

The goal of this project is to study the aspects of immune response in both pouch young (PY) and adult possums that could be important for implementing biocontrol.

The project was undertaken in two contexts - (1) Responses to antigen when adjuvant, especially Freund's is not used. (2) The need to identify aspects of the life history of possums which could be valuable for biocontrol.

Approach & Outcomes

The methodology for the first objective involved immunisation of possums with myobacterial protein MPT64 or a plasmid, which expressed its gene i.e. it was DNA mediated immunisation. Responses were studied using the enzyme Linked Immuno Absorbant assay ELISA. The methodology for the second objective investigation involved the Reverse Transcription Polymerase Chain Reaction (RT-PCR). The technique was applied to RNA extracted from possum's 10-103 days old.

There were three outcomes:

1. Further work on immunocontraception should be aimed at showing that effective immunisation against relevant reproductive antigens can be achieved in the absence of adjuvant, especially Freund's.

2. Investigation of the possibility of inhibiting the transfer of material IgG into the milk or from the pouch young into its blood should be carried out, with the intention of causing early loss of the pouch young through infection by pouch biota.

3. A major outcome was the manufacture of antisera to IgG, IgM, IgA, and IgE, which are now commercially available for studies that require assay of possum immunoglobulins.

Publications

Belov, K., Harrison, G.A., Miller, R.D., Cooper, D.W. (1999): Isolation and sequence of a cDNA coding for the heavy chain constant reion of IgG from the Australian brushtail possum, Trichosurus vulpecula. Molecular Immunology, 36, 534-541.

Belov, K., Harrison, G.A., Rosenberg, G.H., Miller R.D., Cooper, D.W. (1999): Isolation and comparison of the IgM heavy chain constant regions from Australian (Trichosurus vulpecula) and American (Monodelphis domestica) marsupials. Development and Comparative Immunology, 23(7-8), 649-56.

Belov, K., Harrison, G.A., Miller, R.D., Cooper, D.W. (1999): Cloning and characterisation of the Trichosurus vulpecula heavy chain constant regions of IgG, IgM, IgA and IgE. Advances in the biological control of possums, 56, 47-49.

Belov, K., Harrison, G.A., Miller, R.D., Cooper, D.W. (1999): Molecular cloning of the brushtail possum (Trichosurus vulpecula) immunglobulin E heavy chain constant region. Molecular Immunology 36 (18): 1255-61.

Miller, R.D. and Belov, K. (2000): Immunoglobulin genetics in marsupials. Developmental and Comparative Immunology 24 (5): 485-90.

Belov, K., Harrison, G.A., Miller, R.D., Cooper, D.W. (2000): Characterisation of the kappa light chain of the brushtail possum (Trichosurus vulpecula). Veterinary Immunology and Immunopathology 78 (3-4): 317-24.

Belov, K., Harrison, G.A., Miller, R.D., Cooper, D.W. (2000): Molecular cloning of four lamba light chain cDNAs from the Australian brushtail possum (Trichosurus vulpecula). European Journal of Immunogenetics - in press.

Lam, M.K-P., Belov, K., Harrison, G.A., Cooper, D.W. (2000): cDNA Cloning of the MHC Class II DRB gene from the Brushtail possum (Trichosurus vulpecula). Immunology Letters 76 (1): 31-6.

Lam, M.K-P., Belov, K., Harrison, G.A., Cooper, D.W. (2000): cDNA Cloning of the MHC Class I gene from the Brushtail possum (Trichosurus vulpecula). Immunogenetics - in press.

Cooper, D.W., Lam, M.K-P., Corner, L.A., Cowan, P.E., Rawson, R (2001): Lack of association between field acquired bovine tuberulosis and an MHC marker in two population of New Zealand brushtail possums (Trichosurus vulpecula). In preparation.

Lam, M.K-P., Hickson, R.E., Cowan, P.E., Cooper, D.W. (2000): A major histocompatibility (MHC) microsatellite locus in brushtail possums (Trichosurus vulpecula). Online Journal of Beterinary Research 4: 139-141.

Nguyen, M-A, Belov K., Zenger, K.R., Cooper, D.W. (2001): Onset of expression of immunoglobilin genes during pouch young development. Honours thesis, Macquarie University.

5.4 PBC 260

Programme Title: Population and behavioural responses of wild possums to fertility control
Programme Leader: Dr Phil Cowan
Institution: Landcare Research, Palmerston North

Summary

Demographic responses to fertility control

Population abundance has shown no strong upward or downward trend on any sites except the two 80 percent sterility sites, with one declining by an average of 9 percent per year and the other increasing by an average of 19 percent per year. The relative contribution of local recruitment (female recruits born to resident females) and immigration (female yearlings) to the population growth rate revealed that sterilising 50 percent and 80 percent of females reduced local recruitment by an average of 81 percent and 82 percent, respectively. Immigration almost completely compensated for reduced local recruitment, and this explained the positive population growth on one 80 percent sterility site. Population modelling revealed that both 50 percent and 80 percent sterility treatments were effective at restricting the rate of population recovery following a conventional control operation where 80 percent of the population were killed 2 years after application of sterility treatments.

Behavioural responses to fertility control

The transmission rate of L. balcanica infection was significantly lower in possums subject to gonadectomies than intact possums, with a 78 percent reduction in the relative risk of sero-conversion. Castrated male possums had a significantly smaller home range size following gonadectomy treatment. Although females also had a smaller range size following gonadectomy, the difference was not significant. Thus, changes in behaviour may affect rates of contact between individuals which, in turn, may affect disease (Tb) or vector transmission rates.

Background

This research assesses the effects of fertility control on wild possum populations and is an essential step in validating biological control of possums by manipulating fertility. The results of these field experiments will be applicable to a wide range of methods proposed for biological control of possum fertility. This report details progress made during 2000/01.

Approach & Outcomes

Demographic responses to fertility control

The effect of fertility control on the population dynamics of possums is being investigated on six 14-ha study sites. Treatments, consisting of 0 percent, 50 percent or 80 percent of resident females sterilised at a particular site, were applied in early 1996 and adjustments to maintain the levels of sterility were made annually on recruits. Population parameters were estimated and compared between sterilisation treatments. Population modelling was used to assess the potential effects of sterility treatments over time scales longer than the current experiment.

Behavioural responses to fertility control

The effect of disrupting endocrine control of reproduction on individual home ranges and movements was undertaken using surgical gonadectomy on male and female possums. The effect of gonadectomy on the transmission rates of a commonly occurring disease in possums, Leptospira balcanica, was determined from bimonthly serum sampling.

5.5 PBC 265

Programme Title: Synchronised oestrus and controlled mating in possums and oral delivery of biocontrol agents
Programme Leader: Dr Bernie McLeod
Institution: AgResearch

Summary

Objective 1: Controlling follicle populations. To assess the effectiveness of manipulating gonadotrophin secretion in controlling the emergence and development of the preovulatory follicle

In naturally cycling animals, ovulation, oestrus and mating are all initiated by progressively increasing oestradiol concentrations produced by the growing preovulatory follicle immediately before it ovulates. A successful oestrus synchronisation procedure must synchronise the emergence and development of the preovulatory follicle among treated animals. Although the administration of exogenous progesterone will block ovulation in possums, there is poor synchrony of the time of emergence of the preovulatory follicle when progesterone treatment is withdrawn. This variation is 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.

In recent experiments, we have treated animals with a bolus injection of oestradiol immediately prior to an extended period of progesterone administration, to induce the regression of all of the antral follicles present and then suppress further antral follicle development. This combination steroid hormone treatment has been successful in synchronising the emergence of preovulatory follicles, with 80 percent of treated animals having a large (5-6mm diameter) follicle present on the same day (Day 7 after the end of treatment). However, it remains to be determined whether these follicles will continue to develop normally and if so, what degree of synchronisation there will be in the time of ovulation.

Our original strategy was to investigate whether prolactin had a suppressive effect on follicle development in possums, as it does in a number of species. For example, it is well documented that hyperprolactinaemia is associated with ovarian failure; that the suckling stimulus promotes prolactin secretion and blocks ovulation; and that seasonal anoestrus in short-day breeding animals is associated with elevated prolactin concentrations. However, we now question the likelihood of prolactin having similar actions in the possum. For example, a large proportion of possums (e.g. 50 percent in Otago) undergo oestrous cycles in spring, presumably despite rising plasma prolactin concentrations at this time of year. In addition, not all animals will cycle following pouch young removal, which presumably induces a sudden decline in plasma prolactin concentrations (as it does all other species studies). Most importantly, our recent findings of the presence of large numbers of prolactin receptors in the possum ovary suggest an important, and perhaps even unique role for this hormone in ovarian function in this species. Therefore, we are also investigating whether prolactin promotes follicle development and ovulation.

Objective 2: Induced ovulation of mature preovulatory follicles. To assess the effectiveness of hormonal treatments to induce the synchronous ovulation of either spontaneously developing or induced preovulatory follicles.

The strategy is to synchronise the time of ovulation in groups of possums by artificially inducing the rupture of preovulatory follicles. In naturally cycling females, ovulation is initiated by the preovulatory LH surge, which in turn is induced by progressively increasing oestradiol concentrations. The basis of this objective is to determine whether exogenous oestradiol or LH (or analogues of LH such as hCG) will induce ovulation of preovulatory follicles in possums.

The high oestradiol concentrations associated with ovulation are also responsible for making the female receptive to the male so that mating can occur. A critical characteristic of any synchronisation procedure is that mating should occur. This objective will not only evaluate hormonal methods for their ability to induce ovulation, but will also determine whether they are associated with the expression of overt oestrus.

Objective 3: Permeation enhancers, metabolic inhibitors and peptide uptake. To develop formulations incorporating permeation enhancers and metabolic inhibitors that will promote the uptake of peptides across the possum gut wall

In this objective, we are utilising a surgically prepared in situ animal model that we have developed, to assess absorption of compounds across the gastrointestinal wall of the possum. We have used this experimental model to perfuse solutions containing a test material at a known concentration, at a predetermined rate through a measured section of gut. This allowed uptake of the test material within a specific region of the gastrointestinal tract to be quantified, with absorption being determined on the basis of concentrations measured in the peripheral circulation.

In a series of experiments using this in situ model, we identified permeation enhancers that promote absorption rates of low molecular weight peptides across epithelial mucosa of the possum hindgut. These experiments involved constant perfusion of the test material through a section of gut from which the luminal contents had been removed. There are two primary aims of this objective. Firstly, to assess the effects that the presence of luminal contents will have on the rate of absorption and, using information from earlier in vitro studies, determine requirements for metabolic inhibitors within the formulation to maximise uptake. The second aim is to establish whether there are limitations in the size of peptide that can be absorbed under these conditions, by using peptides of increasing molecular weight.

Objective 4: Targeted delivery of peptides and proteins to the possum colon and caecum. To identify polymer coatings and delivery systems that will protect formulations containing biocontrol agents from degradation in the stomach and small intestine and release them in the possum hindgut.

The very large caecum and colon of the possum are likely to be the target sites for uptake of biocontrol agents across the gut wall. These regions are main fermentation sites so ingested compounds will have a long residence time in there, and we have shown that the rate of degradation of peptides and proteins is many times lower than in the small intestine. Therefore, a biocontrol agent must be formulated in such a way that it is protected from degradation in the acid environment of the stomach, protected from proteolytic activity of luminal enzymes within the small intestine, then released after reaching the hindgut. In clinical medicine, enteric coatings that are insoluble at low pH are frequently used to protect bioactive compounds from degradation in the stomach and these are likely to be similarly effective in the possum. However, cost will be an important issue to be considered for delivery to possums. Different strategies are employed to maintain protection while in transit through the small intestine and to ensure release in the caecum and colon, as there are only small differences in pH between the duodenum, jejunum, ileum, caecum, proximal colon and distal colon. These can be based on timed release, where the time taken for the polymer coating to disintegrate is longer than the transit time from mouth to hindgut, or on the use of compounds that are degraded by the micro-organism population of the hindgut, not present in the small intestine. Either approach requires the environment of each region of the gastrointestinal tract to be characterised.

Background

The goal of this project is 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.

Approach & Outcomes

Objectives 1 and 2

The aim of this project is to synchronise the time of oestrus, ovulation and mating in possums. Over her 26-day oestrous cycle, a female possum will develop a single preovulatory follicle that will eventually ovulate to release its egg, and if the egg is fertilised, she will invariably produce a single young. Oestrogen, produced by the preovulatory follicle prior to its rupture, induces both oestrus and ovulation, thus making the female receptive to males at a time that maximises the chance of the egg being fertilised. Within a population of breeding female possums, these events will be occurring randomly, so each female will ovulate at some time over a 26-day period. The effectiveness and reliability of any possum biocontrol method will be measured as the percentage of fertile females in which reproduction is disrupted. To realistically determine this, reproductive success will need to be monitored in groups of females at the same time. This will require access to large groups of female possums in which the time of ovulation and mating is synchronised.

There are numerous reports of how treatment with exogenous hormones can be used to influence follicle development and induce superovulation in possums. However, all of these treatments frequently lead to abnormal ovarian function. None of them consistently result in the ovulation of a single follicle, are associated with the expression of oestrus and mating or with successful pregnancies.

In the past, we have used standard hormonal treatments that are used successfully in other animals to synchronise oestrus and ovulation in possums. Typically, these involve synchronising the onset of the follicular phase of the oestrous cycle. By synchronously removing a hormonal block on the development of large antral follicles, the progressive growth and ovulation of follicles is synchronised between treated animals. These methods fail in the possum because of the very long, and highly variable (8-14 days), follicular phase in this species. Although the onset of follicle development may be synchronised, its rate of development, and thus the time of ovulation, is highly variable.

The approach we now take in attempting to synchronise oestrus and ovulation in possums is in two parts. Firstly, the aim is to synchronise the emergence of the preovulatory follicle and then artificially induce its ovulation to overcome the inherent asynchrony between animals. The end-point of this programme will be the ability to induce the synchronised development of a single preovulatory follicle without incurring any abnormal follicle development, followed by the ovulation (within a 24h period between animals) of that follicle, leading to successful pregnancy and the birth of a live young.

Objectives 3 and 4

Current research into new biocontrol technologies for possum control is wide ranging and many different molecules are being investigated as potential vaccines, antigens or toxins that might interfere with possum biology. A critical part of any novel biocontrol method will be the delivery of such molecules to the possum. In the short to medium-term, it is likely that delivery in the form of a bait (oral delivery) will be important. For any biocontrol agent that is to be delivered via the oral route, there will be problems of stability during manufacture and storage, during dispersal in the field and during their passage in the possum gastrointestinal tract that will need to be resolved. In addition, the biocontrol agents will need to be formulated in such a way that they are initially protected from degradation, and eventually released at a specified site and with the desired profile, to maximise their uptake in the appropriate region of the gut and therefore to maximise their biological effect. These two objectives relate to collaborative research between AgResearch Invermay and the School of Pharmacy, University of Otago, taking a `pharmaceutical science' approach to developing oral delivery in the possum.

Publications

McLeod BJ, Thompson EG, Mathieson, S, Tucker IG, Davies NG, Ledger R, Wen J, Zhang H, Butt AG. 2000. Oral delivery of biocontrol agents - solving the problems of targeted delivery in possums. In; Possum & Bovine Tb management in 2010. NSSC Workshop, Wellington.

McLeod BJ, Tucker IG. 2000. Designer drugs for possums. AgScience 1, 7-8.

Tucker IG, Butt AG, McLeod BJ. (2001) Delivery of bioactives to possums. National Science Strategy Workshop Wellington.

McLeod BJ, Wen JY, Davies NM, Butt AG, Ledger R, Tucker IG. (2001) Oral delivery to possums - protecting bioactives and enhancing their uptake. National Science Strategy Workshop Wellington.

Butt AG, Wen J, Tucker IG, Davies N, Ledger R, McLeod BJ. (2001) The possum intestine - a barrier and a target. National Science Strategy Workshop Wellington.

Eckery DC, Juengel JL, Bauer E, Heath DA, Lawrence SB, Lun S, McLeod BJ, Moore LG, Thompson EG, Thomson BP, Western A, Whale L, McNatty KP. (2001) Disabling reproductive function in possums; research findings and future directions. National Science Strategy Workshop Wellington.

Margot Skinner, Neil Wedlock, Elizabeth Doolin, Keith Hamel, Trish Mahoney, Bernie McLeod and Bryce Buddle. (2001) Stimulation of mucosal immune responses in possums National Science Strategy Workshop Wellington.

McLeod BJ, Thompson EG. (2001) Predation on sparrows by possums in captivity. Notornis (submitted)

Wen JY, Davies NM, Ledger R, Butt AG, McLeod BJ, Tucker IG. (2001) Determination of the in vitro degradation of Luteinising Hormone Releasing Hormone in possum gut by high performance liquid chromatography and amino acid analysis Journal of Chromatography (submitted)

Wen JY, Ledger R, Davies NM, Butt AG, McLeod BJ, Tucker IG. (2001) Metabolism of BSA and LHRH by luminal contents and mucosal homogenates from the gastrointestinal tract of the brushtail possum (Trichosurus vulpecula). Journal of Comparative Biochemistry and Physiology B (submitted)

Wen JY, Butt AG, Davies NM, Ledger R, McLeod BJ, Tucker IG. (2000) Enzyme barriers to intestinal absorption of peptides and antigens in the possum. Formulation and Drug Delivery Conference Dunedin.

Wen JY, Butt AG, Davies NM, McLeod BJ, Tucker IG. (2000) The permeability properties of the large intestine of the brushtail possum. Formulation and Drug Delivery Conference Dunedin.

Legge M, Homer C and McLeod BJ. (2000). Role of the adrenal gland special zone in steroidogenesis in the reproductive cycle of Trichosurus vulpecula. Annual Conference of the Society for the Study of Fertility. Edinburgh.

Butt AG, McLeod BJ. (2000) Variations in dietary Na intake do not modify amiloride sensitive Na transport in the colon of the brushtail possum. Annual Conference of the New Zealand Physiological Society Auckland

McLeod BJ, Thompson EG, Mathieson S, Tucker IG, Davies NM, Ledger R, Wen JY, Zhang H, Butt AG. (2000) Oral delivery of biocontrol agents to possums - solving the problems of targeted delivery in possums. National Science Strategy Workshop Wellington.

McLeod BJ. (2000) Oral delivery of biocontrol agents, new findings and implications for use of conventional toxins. National Possum Control Agencies National Technology Transfer Seminar Wellington.

Wen JY, Davies NM, Butt AG, Ledger R, McLeod BJ, Tucker IG. (2001) Issues in the Oral Delivery of Peptides to Possums: The Physical Barrier. Formulation and Delivery of Bioactives Conference Dunedin

Tucker IG, Butt AG, McLeod BJ. (2001) Issues in oral delivery - getting bioactives into and through the possum gut. National Science Strategy Workshop Wellington.

McLeod BJ, Wen JY, Davies NM, Butt AG, Ledger R, Tucker IG. (2001) Oral delivery to possums -protecting bioactives and enhancing their uptake. National Science Strategy Workshop Wellington.

Eckery DC, Juengel JL, Bauer E, Heath DA, Lawrence SB, Lun S, McLeod BJ, Moore LG, Thompson EG, Thomson BP, Western A, Whale L, McNatty KP. (2001) Disabling reproductive function in possums; research findings and future directions. National Science Strategy Workshop Wellington.

Margot Skinner, Neil Wedlock, Elizabeth Doolin, Keith Hamel, Trish Mahoney, Bernie McLeod and Bryce Buddle. (2001) Stimulation of mucosal immune responses in possums National Science Strategy Workshop Wellington.

Wen JY, Davies NM, Butt AG, Ledger R, McLeod BJ, Tucker IG. (2001) Issues in oral delivery of proteins and peptides to possums I. Enzymatic Barriers Australian Pharmaceutical Science Association Newcastle.

Mahoney PM, Hurst PR, McLeod BJ, McConnell MA. (2001) Are mast cells important for reproductive function in the brushtail possum? Annual Conference of the Society for the Study of Fertility. Cambridge.

Mahoney PM, Hurst PR, McLeod BJ. (2001) Morphology of mast cells in the vaginal cul-de-sac of the brushtail possum. Annual Conference of Australian Society of Reproductive Biology. Gold Coast

5.6 PBC 266

Programme Title: Development of stably-transfected cell lines for GnRH-R and a possum-specific assay
Programme Leader: Dr Ken McNatty (formerly Dr Jerome Demmer)
Institution: AgResearch

Summary

In January, due to the departure of Dr. Jerome Demmer, the milestones for this programme were revised. This was done to take advantage of the current skill base at Wallaceville and to add greater effort to understanding the role of prolactin (Prl) in possum reproduction and to aid in the development of better GnRH-toxin conjugates.

Prolactin is an important hormonal regulator of lactation in many species including marsupials. We have identified receptors for Prl in the possum mammary gland and also in various cells types within the possum ovary. Interestingly, the findings in the ovary are different from other mammals and suggest that Prl may play an important role in ovarian function. To help determine the role of Prl in possum reproduction we have produced recombinant possum (rcpos) Prl and are developing a Prl radioimmunoassay.

Background

The goal of this project is to determine the classes and quantities of immunoglobulins in the milk and to characterise the mechanism required to transport them into milk for the development of methods for the biological control of possums.

Approach & Outcomes

Initial efforts to purify Prl from possums were unsuccessful, however we had cloned the gene for Prl and so it was possible to produce the large amounts of Prl we needed using molecular biology techniques. We have produced rcposPrl and two Prl mutant proteins from E. coli. We have shown using an in vitro bioassay that the rcposPrl is biologically active, having similar activity to native sheep Prl. In addition, we found that the two mutant proteins were able to bind to the Prl receptor but did not stimulate a signal transduction pathway and were able to block the binding of normal Prl. These results suggest that two new Prl antagonists have been produced. These recombinant proteins will be very valuable tools in our research programme.

Biologically active rcposPrl was further purified by FPLC and used to immunise rabbits. The antibodies produced were able to recognise rcposPrl and two different preparations of native sheep Prl; suggesting that the recombinant protein was able to refold properly into its native form. Further assessments of these antibodies and development of a Prl radioimmunoassay are currently underway.

One of the goals of this programme is to develop a stable cell line expressing the possum GnRH receptor. This cell line will be used for screening the bindability and evaluating the toxicity of different GnRH-toxins. Several clones have been produced that express the GnRH receptor, however expression has always been either unstable or very low. To overcome this, we have designed a new expression plasmid construct. Sequencing revealed that the 5'UTR region that was put into the construct to increase expression had recombined resulting in a truncated form. Work is continuing to resolve this aberration and successfully establish a cell line expressing the possum GnRH receptor.

5.7 PBC 268

Programme Title: Riboflavin carrier protein (RCP): Identification, purification and biological consequences
Programme Leader: Ken McNatty
Institution: AgResearch

Summary

Immunisation of possums against cRCP increased the time it took for possums to become pregnant, but it did not affect the number of possums that produced offspring. However, possums showed a poor immune response to cRCP. In a study designed to increase the immune response, possums were immunised against cRCP coupled to keyhole limpet haemacyanin, which is known to be highly immunogenic in possums. This did not increase the immune response, but only 1 of 5 possums immunised produced offspring. These results were encouraging and provided some evidence that immunisation against RCP could adversely affect fertility in possums. However, all the work investigating the effects of RCP on fertility in other species was reported primarily by one laboratory and before extensive fertility trials were carried out in possums we wanted to confirm the anti-fertility effects that had been reported in mice. Unfortunately, we were unable to show any effects of passive or active immunisation on the fertility of mice. Thus, further work in possums was not carried out.

Despite an extensive amount of work, we have not been able to purify RCP from a range of mammalian species nor have we been successful at identifying the gene for RCP in possums or mice. Although there was some indication that immunising possums against cRCP could affect fertility, we were unable to confirm the ant-fertility effects that have been reported in mice, and therefore additional work in possums could not be justified and was not carried out. Although RCP initially seemed to be a good candidate for possum control, it is clearly not and no further work is proposed in this area.

Background

The goal of this work was to evaluate riboflavin carrier protein (RCP) as a potential target for the immunosterilisation of possums. RCP enables riboflavin (vitamin B2) to cross cellular barriers and is thought to be essential for the survival of developing embryos and for delivering riboflavin to sperm. Immunisation of female rats, mice and monkeys against chicken (c)RCP has been shown to cause early embryonic loss. Immunisation of male rats and monkeys against cRCP has also been shown to cause infertility. In addition, 21-residue synthetic peptide epitopes from cRCP have been used successfully to interfere with reproductive function in rodents and monkeys. Nevertheless, even though RCP seems to be essential for fertility in mammals, only small amounts of RCP have been reported to be purified or partially purified and the sequence of RCP is not known for any mammalian species. Thus, the aims of this work were to purify and determine the sequence for possum RCP, identify the gene for RCP and determine the effects of immunisation against RCP in possums.

Approach & Outcomes

In order to purify RCP from possums we first needed some way of detecting whether it was present or not. For this, we produced antibodies against cRCP in rabbits and developed a radioimmunoassay. Many attempts using several methods were made at purifying RCP from possum tissues and biological fluids without success; these included liver, kidney, heart, mammary, uterus, spleen, pancreas, brain, serum and amniotic fluid. Attempts were also made at purifying RCP from other species including mice, cows, sheep and deer, but were also unsuccessful. Some RCP activity was detected in liver and uterus of pregnant mice, but Western blots of the same tissues gave negative results suggesting that if RCP was present it was in such low quantities that purification was not feasible. In contrast, RCP was easily detected and purified from chicken eggs. Thus, to our knowledge RCP has not been successfully purified from any mammalian species.

Because the purification of RCP was so difficult we decided to try to isolate the gene and obtain the sequence for RCP through molecular biology. We initially used an RT-PCR based method to amplify possum RCP cDNA. Twenty-five different primers were used, but all attempts were unsuccessful. We then chose a genomic DNA approach because it does not rely on the same sort of primer specificity as RT-PCR or a knowledge of the tissues where the gene is expressed. Southern blots were done on genomic DNA isolated from both possums and mice and a possum genomic library was screened using a cRCP probe. All results were negative. In addition, sequence databases (e.g. AgResearch bovine and ovine EST databases and those available on the internet) have been checked regularly for sequences that are homologous to cRCP, but none have been found.

5.8 PBC 269

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

Summary

A major goal of our research programme is to devise methods to disrupt the actions of key reproductive hormones. One method we are investigating is the use of the hypothalamic peptide hormone, gonadotrophin-releasing hormone (GnRH), as a vehicle to deliver toxins specifically to gonadotrophin-secreting cells in the pituitary gland. GnRH-toxin conjugates act directly on target cells and therefore provide an alternative approach to fertility control that does not rely on the immune system. The premise for the use of GnRH-toxin conjugates is that after GnRH is bound to specific receptors on pituitary cells, it becomes internalised into those cells, thereby delivering the toxin only to those cells that produce reproductive hormones. In order for this technology to work in possums, it was necessary to establish the role of GnRH in pituitary function, the sites of GnRH action, the rate of GnRH internalisation and whether toxins could be introduced into cells by this method. We have previously reported that GnRH is indeed a primary regulator of FSH and LH secretion in possums, and that the actions of GnRH are exclusive to the pituitary gland via specific high affinity receptors; suggesting that the use of GnRH-toxins has the potential to disrupt the actions of reproductive hormones while ensuring organ specificity.

The rate at which GnRH is internalised and the properties of the chemical linkage between GnRH and the toxin will have significant implications on the doses of GnRH-toxins required and their effectiveness. Using an in vitro culture system, we have determined that internalisation of GnRH into possum pituitary cells occurs within 15 minutes of binding and that maximum internalisation occurs within 30 minutes This rapid internalisation should allow toxins to get into pituitary cells quickly following binding of GnRH.

We have investigated the effects of two GnRH-toxin conjugates, namely GnRH-PAP (pokeweed antiviral protein) and GnRH-RNase, both in vitro and in vivo. In cultured possum pituitary cells treated overnight, GnRH-PAP caused a significant decrease in LH secretion and in the number of cells containing LH, suggesting that the toxin was able to enter cells and kill them. GnRH-PAP was not effective at doses less than 1ng/ml or when exposure was only for four hours. GnRH-RNase was not effective at any dose or time tested. RNase was chosen as a toxin for testing because it is smaller than PAP and also has the possibility of being species specific. The same GnRH-RNase conjugate used in these experiments was found to be effective in other species, including cats and sheep, suggesting that there may be differences in the types of RNase that may be effective in possum cells.

One of the difficulties we have faced in testing GnRH-toxins in vitro is that GnRH on its own causes a downregulation of gonadotrophic cells. To overcome this difficulty and to aid in the evaluation of GnRH-toxins in general, we have attempted to establish a stable cell line expressing the possum GnRH receptor. To date we have been unsuccessful. Several clones have been produced that express GnRH receptor, however expression has either been unstable or very low. New plasmid constructs have been made and will be tested in the future.

Studies have been done with limited amounts of reagents to test the effects of GnRH-PAP and GnRH-RNase in vivo. Results were similar to in vitro studies, where GnRH-PAP showed some ability to reduce the secretion of gonadotrophins whereas GnRH-RNase showed no effects. The effects of GnRH-PAP were only temporary and gonadotrophin levels returned to normal within three weeks of treatment. Nevertheless, these results did show that GnRH-PAP can affect circulating levels of gonadotrophins in possums and provided evidence that toxins can be introduced into cells by this method.

Overall we have established the feasibility for the use of GnRH-toxins in possums and future studies will be focused on determining their effects in vivo and also identifying new toxins that may be used in this technology.

Background

The goal of this project was to control reproduction by targeting the possum pituitary gland through the use of GnRH-toxin conjugates.

Approach & Outcomes

The premise for the use of GnRH-toxin conjugates is that after GnRH is bound to specific membrane receptors on pituitary cells, the GnRH-receptor complex will be internalised thereby delivering the toxins only to those cells that produce reproductive hormones. The rate at which the receptor-ligand complex is internalised will have profound effects on the does of GnRH-toxin needed and the type of chemical bond used for conjugation. For example, 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, thereby preventing the toxin from entering into the cell.

5.9 PBC 267

Programme Title: Immunology of bovine tuberculosis
Programme Leader: Dr Bryce Buddle
Institution: AgResearch

Summary

  • Vaccination of cattle with mycobacterial DNA vaccines induced weak cellular immune responses to mycobacterial antigens, but no protection against challenge with virulent M. bovis. Revaccination of DNA-vaccinated cattle with a mycobacterial protein in an oil adjuvant did not enhance protective immunity.
  • Real-time PCR analysis was used to quantify the levels of antigen specific mRNA expression of two cytokines, IFN-_ and IL-4. The BCG-vaccinated cattle which expressed the highest level of IFN-_ mRNA expression also expressed the highest level of IL-4 mRNA. Following challenge, expression of IL-4 mRNA was correlated with severity of disease. BCG vaccination induced antigen specific activation of CD4 and CD8 T cell subsets.
  • A technique has been established for cannulating the efferent lymph duct of the prescapular lymph node. This will allow for studies on regional immunity when cattle are immunised with tuberculosis vaccines in the neck region.
  • A combination of two M. bovis specific antigens, ESAT-6 and CFP10 greatly enhanced the specificity of the IFN-_ test for the diagnosis of bovine tuberculosis compared to standard IFN-_ test, yet still maintained a very high level of sensitivity. This new test has great potential to reduce false positive reactions. The addition of recombinant IL-2 to the whole blood cultures did not enhance the sensitivity or specificity of the IFN-_ test.
  • Possums vaccinated intranasally and intraconjunctivally with two new attenuated M. bovis vaccines had a greater level of protection against an aerosol challenge with M. bovis than following vaccination with BCG. Interestingly, attenuated vaccines, which induced high levels of protective immunity when used subcutaneously, did not necessarily induce equivalent levels of immunity when administered via intranasal and conjunctival routes.
  • In a AHB-funded project, an encapsulated BCG vaccine administered orally to possums induced significant protection against an aerosol challenge with M. bovis.
  • A vaccination strategy based on priming with a DNA vaccine and boosting with live attenuated M. bovis strains induced a higher number of antigen specific IFN-_ secreting cells from mouse spleens than that induced by vaccination with the attenuated M. bovis strains alone. However, there was no enhancement in the level of protection of mice against an aerosol challenge with virulent M. bovis.
  • Antibodies are now available to phenotype possum B and T lymphocytes. Considerable progress has been made in the cloning and expression of a possum cytokine TGF_ that could be useful to enhance mucosal antibody responses for the development of immunosterilising vaccines.
  • The intranasal administration of either recombinant TNF-_ or BCG with a model protein antigen promoted antibody responses to the model antigen.
  • Immunohistochemical methods have been developed for staining possum B and T cells in Peyers patches.
  • Possums immunised intranasally at oestrus had different levels of antibodies in serum and mucosal secretions than those immunised 12 days post oestrus.
  • Seventeen papers were published or submitted to refereed journals in the 2000/01 year.

Background

The goal of this project was to improve diagnostic tests and vaccines for control of bovine tuberculosis.

Approach & Outcomes

Development and evaluation of diagnostic tests and vaccines for control of bovine tuberculosis in cattle and possums.

  • Determine whether DNA tuberculosis vaccines can protect cattle against bovine tuberculosis.
  • Determine the activation and cytokine profiles of different T-cell subsets after vaccination with BCG.
  • Establish a technique for the cannulation of the efferent lymph duct of the prescapular lymph node of calves to allow the study of the regional immunity to tuberculosis vaccination.
  • Improve the specificity of the IFN-_ test for the diagnosis of bovine tuberculosis by using a range of different M. bovis specific antigens and by adding recombinant IL-2 to the whole blood cultures.
  • Establish whether four newly attenuated M. bovis strains administered as vaccines can protect possums against bovine tuberculosis better than BCG.
  • Compare different types and doses of anti-ulcer drugs and antacids for reducing gastric acidity in possums to enhance the efficacy of an orally administered BCG vaccine.
  • Determine the efficacy of a vaccination strategy based on priming with a DNA vaccine and boosting with live attenuated M. bovis strain compared to vaccination with either of the two components alone using a mouse tuberculosis model.
  • Submit three papers to refereed journals.
  • Develop methods for measuring mucosal immune responses in possums. Develop vaccine delivery systems and further our understanding of mucosal immunity in possums.
  • Develop reagents for phenotyping possum lymphocytes and isolate one new possum cytokine.
  • Determine which adjuvants and vaccine formulations, administered by the intra-nasal route, stimulate high levels of antibodies in the mucosal secretions of possums and use the best to test for mucosal immune responses after oral administration.
  • Develop immunohistochemical methods to measure local antibody secreting cells at mucosal sites and determine changes in the type of antibody produced after immunisation with different vaccines.
  • Determine whether antibody levels in reproductive secretions of female possums are influenced when possums are immunised around the time of oestrus.
  • Submit one paper to a refereed journal.

Publications

Pollock, J.M., Girvin, R.M., Lightbody, K.A., Clements, R.A., Neill, S.D., Buddle, B.M. and Andersen, P. 2000. Assessment of defined antigens for the diagnosis of bovine tuberculosis in skin test-reactor cattle. Vet Rec. 146: 659-665.

Ryan, T.J., Buddle, B.M. and de Lisle, G.W. 2000. An evaluation of the gamma interferon test for detecting bovine tuberculosis in cattle 8 to 28 days after tuberculin testing. Res. Vet. Sci. 69, 57-61.

Wedlock, D.N., Vesosky, B., Skinner, M.A., de Lisle, G.W., Orme, I.M. and Buddle, B.M. 2000. Vaccination of cattle with Mycobacterium bovis culture filtrate proteins and interleukin-2 against bovine tuberculosis. Infect. Immun. 68: 5809-5815.

Skinner, M.A., Prestidge, R., Yuan, S., Strabala, T.J. and Tan, P.L.J. 2001. The ability of heat-killed Mycobacterium vaccae to stimulate a cytotoxic T cell response to an unrelated protein is associated with a 65kDa heat shock protein. Immunology 102: 225-233.

Buddle, B.M. 2001. Vaccination of cattle against Mycobacterium bovis. Tuberculosis. 81:125-132.

Pollock, J.M., Buddle B.M. and Andersen, P. 2001. Towards more accurate diagnosis of bovine tuberculosis using defined antigens. Tuberculosis. 81: 65-69.

Aldwell, F.E., Wedlock, D.N., Slobbe, L.J., Griffin, J.F.T., Buddle, B.M. and Buchan, G.S. 2001. In vitro control of Mycobacterium bovis by macrophages. Tuberculosis. 81: 115-123.

Buddle, B.M., Ryan, T.J., Pollock, J.M., Andersen, P. and de Lisle, G.W. 2001. Use of ESAT-6 in the interferon-_ test for diagnosis of bovine tuberculosis following skin testing. Vet. Microbiol. 80: 37-46.

Skinner, M.A., Wedlock, D.N. and Buddle, B.M. 2001. Vaccination of animals against Mycobacterium bovis. Rev. Sci. Tech. Off. Int. Epiz. 20 (1): 112-132.

Doolin, E.E., Midwinter, R.G., McCarthy, A.R., and Buddle, B.M. 2001. Purification of secretory immunoglobulin A from milk of the Australian brushtail possum (Trichosurus vulpecula). N.Z. Vet. J. (in press).

Corner, L.A., Buddle, B.M., Pfeiffer, D.U. and Morris, R.S. 2001. Aerosol vaccination of the brushtail possum (Trichosurus vulpecula) with bacille Calmette-Guérin: the duration of protection. Veterinary Microbiology. (in press).

Wedlock, D.N., Skinner, M.A., de Lisle, G.W. and Buddle, B.M. 2001. Control of Mycobacterium bovis infections and the risks to human populations. Microbes & Infection (submitted).

Buddle, B.M., Wards, B.J., Aldwell, F.E., Collins, D.M. and de Lisle, G.W. 2001. Influence of sensitisation to environmental mycobacteria on subsequent vaccination against bovine tuberculosis. Vaccine (submitted).

Skinner, M.A., Keen, D.L., Parlane, N.A., Yates, G.F. and Buddle, B.M. 2001. Increased protection against bovine tuberculosis in the brushtail possum (Trichosurus vulpecula) when BCG is administered with killed Mycobacterium vaccae. Tuberculosis (submitted)

Corner, L.A., Buddle, B.M. and Morris, R.S. 2001. Conjunctival vaccination of the brushtail possum (Trichosurus vulpecula) with bacille Calmette Guerin. Wildlife Diseases (submitted).

Corner, L.A., Buddle, B.M., Pfeiffer, D.U. and Morris, R.S. 2001. Vaccination of the brushtail possum (Trichosurus vulpecula) with bacille Calmette-Guérin: the response to multiple doses. Veterinary Microbiology. (submitted).

Corner, L.A., Buddle, B.M., and Morris, R.S. 2001. Infection of the brushtail possum (Trichosurus vulpecula) with bovine tuberculosis by the conjunctival route. The Veterinary Journal (submitted).

Buddle, B.M. Large animal models for vaccination against tuberculosis. TB into the New Millennium. Cairns, Australia, 14-18th July 2000, pp. 66-68.

Corner, L.A., Buddle, B.M., de Lisle, G.W., Norton, S. and Morris, R.S. BCG aerosol vaccination of brushtail possums against Mycobacterium bovis infection in the wild. Salman, M.D., Morley, P.S. & Ruch-Gallie R. Proceedings 9th Symposium of the International Society for Veterinary Epidemiology and Economics. Abstract No. 252.Colorado State University, August 2000.

Skinner, M.A., Keen, D.L., Parlane, N.A. de Lisle G.W., and Buddle, B.M. Heat-killed Mycobacterium vaccae improves the efficacy of M. bovis BCG vaccination in possums. 3rd International Conference on Mycobacterium bovis, Cambridge, UK., 13-16th August 2000.

Ryan, T.J., Griffin, J.F.T., de Lisle G.W., Rogers, C.R., Buddle, B.M. and Livingstone, P.G. Tuberculosis in cattle; introduction of blood tests into the national control program in New Zealand. 3rd International Conference on Mycobacterium bovis, Cambridge, UK., 13-16th August 2000.

Corner, L.A., Buddle, B.M, de Lisle, G.W., Norton, S. and Morris, RS. BCG vaccination of wild brushtail possums. 3rd International Conference on Mycobacterium bovis, Cambridge, UK., 13-16th August 2000.

de Lisle, G.W., Keen, D.L., Parlane, N.A., Yates, G.A., Collins, D.M., and Buddle, B.M. Vaccination with attenuated Mycobacterium bovis strains protects possums against aerosol challenge. 3rd International Conference on Mycobacterium bovis, Cambridge, UK., 13-16th August 2000.

Aldwell, F.E., Wedlock, D.N., Slobbe, L.J., Buddle, B.M., Buchan, G.S., Mackintosh, C.G. and Griffin, J.F.T. Macrophages and M. bovis-victim or victor? 3rd International Conference on Mycobacterium bovis, Cambridge, UK., 13-16th August 2000.

Buddle, B.M. Vaccination of cattle against Mycobacterium bovis. 3rd International Conference on Mycobacterium bovis, Cambridge, UK., 13-16th August 2000.

McNair, J., Girvin, R.M., Clements, R.A., Neil, S.D., Buddle, B.M., Anderson, P. and Pollock J.M. More specific diagnosis of bovine tuberculosis using defined antigens such as ESAT-6. 3rd International Conference on Mycobacterium bovis, Cambridge, UK., 13-16th August 2000.

Skinner, M.A., Rich, F., Buddle, B., Collins, D., de Lisle, G., Ramsey, A., Buchan, G. A prime-boost strategy for vaccination against bovine tuberculosis. 4th Annual Conference on Formulation and Delivery of Bioactives, Dunedin, NZ, 15-16th February 2001.

Rich, F. Wedlock, D.N., Aldwell, F.E. and Buddle, B.M. Identification of differentially expressed genes in Mycobacterium bovis-infected alveolar macrophages by use of suppression subtractive hybridisation. 10th Annual Queenstown Molecular Biology Meeting, Queenstown, 16 -21st August, 2000.

Parlane, N.A., Buddle, B.M., Skinner, M.A. Interleukin 2 receptor expression on subsets of T cells in a DNA vaccine trial in cattle. Australasian Flow Cytometry, Adelaide, Australia, November 2000.

Aldwell, F.E., Slobbe, L.J., Wedlock, D.N., Buddle, B.M. and Buchan, G.S. Macrophages and tuberculosis - victim or victor? Australasian Society for Immunology 30th Annual Conference, Sydney, Australia, December 2000.

Wedlock, D.N., Aldwell, F.E. Rich, R.J. and Buddle, B.M. Identification of differentially expressed genes in Mycobacterium bovis-infected alveolar macrophages by use of suppression subtractive hybridisation. Australasian Society for Immunology 30th Annual Conference, Sydney, Australia, December 2000.

Buddle, B.M. Cattle: the ultimate animal model. Keystone symposia. Molecular and cellular aspects of tuberculosis research in the post genome era. Taos, New Mexico, USA, 25-30th January, 2001.

Skinner, M., Wedlock N., Doolin L., Hamel, K., Mahoney T., McLeod, B, and Buddle B.M. Stimulation of mucocal immune responses in possums. NSSC Possum Biocontrol Workshop, AgResearch, Wallaceville, Upper Hutt, 2-4th April 2001.

Parlane, N.A. Interleukin 2 receptor expression on subsets of T cells in vaccine trials in cattle. New Zealand Flow Cytometry Group Meeting Wellington, April 2001.

5.10 PBV 270

Programme Title: Manipulation of possum viruses
Programme Leader: Tao Zheng
Institution: AgResearch

Summary

  • A sensitive ELISA has been developed for screening possum sera for wallaby herpes virus MaHV-1
  • Twenty-eight possums from an area (Paraparaumu) previously shown to contain possums sero-positive for MaHV-1 were sero-negative.
  • A total of 355 possum sera from two sites in the South Island (Riverton and Golden Bay) were tested and were sero-negative
  • Reactivation of latent herpes virus in 20 sero-negative trapped possums from Paraparumu was attempted using immunocompromised hosts
  • New possum cell lines for the isolation of possum viruses were established and characterised
  • A survey of New Zealand wallabies to screen for additional herpes and other viruses that may be useful in possum biocontrol was started.

Background

The goal of this project is to isolate, characterise and genetically manipulate possum viruses for the biological control of possums.

Viruses can control possum populations in at least two ways; as lethal agents and as disseminating vectors to deliver immunosterilisation vaccines and bioactive molecules.

A herpes virus would be an ideal candidate to serve as a virus vector, as herpes viruses are large viruses that can accommodate foreign genes and are highly transmissible. Many herpes viruses are sexually transmitted and this property could lead to a boost in antibody titres at mating. Another useful feature is that herpes viruses frequently establish latent infections. They are generally easy to propagate in cell culture, which is important for the development of a virus vector. Most animal species have yielded at least one herpes virus upon examination and at least 10 strains of macropodid herpes viruses have been isolated from wallabies and kangaroos in Australia. A herpes virus has been identified by negative stain electron microscopy in two samples of intestinal contents from New Zealand possums (Rice et al., 1996) and serum positive for herpes virus has been found. A serological survey on a possum serum bank collected from 1993-96 led to the identification of possum sero-positive for herpes virus in the areas of Paraparaumu, Wanganui, Orongorongo, Banks Peninsula, Shannon and Kawau Island. In order to isolate possum herpes viruses, it is important to identify areas where there may be sero-positive possums.

Approach & Outcomes

Isolation and characterisation of possum viruses (possum herpes virus) with the aim of developing a viral based foreign gene delivery system for the biocontrol of possums by:

  • Screening fresh collected sera from two sites in the South Island (AgResearch/Landcare joint study) for antibodies to herpes virus.

A number of possums from areas that had sero-postive possums during the 1993-96 period were recently tested and were found to be sero-negative for herpes virus. In particular 28 possums recently trapped in the Paraparumu area were sero-negative. An extensive poisoning programme has been in place in this area since 1993, which may have killed most of the infected possums and reduced the transmission of herpes virus amongst them. In order to identify areas that might currently contain sero-positive possums, antibodies against herpes virus in recent collections of possum serum from two sites in the South Island were measured. In February 2000, 183 possum sera were collected from the Riverton area and in May 2000, 172 sera were collected from the Golden Bay area. So that sera could be screened more effectively an ELISA was developed using a purified antigen from the Wallaby herpes virus MaHV-1. Instead of using crude virus-cell lysate as the coating antigen, virus was purified from the cell lysate. The sensitivity of the assay was increased about 10 times compared to that of the crude antigen. This assay was used for screening possum sera, from the two South Island sites, for antibody against herpes virus and although a more sensitive assay was used no positive sera were identified in these samples. Thirty-nine possum blood samples have been collected from the Orongorongo area, courtesy of Dr Dave Ramsey of Landcare Research and testing of sera is in progress.

  • Investigating the reactivation of possum herpes virus from sero positive possums;

The aim of this objective was to determine whether a possum herpes virus could be isolated from possums by reactivation of latent virus. Although there maybe a greater probability of isolating a possum herpes virus by reactivation of latent virus from tissue of sero-positive possums, no sero-positive possums have been identified from a large number of recently trapped possums. The ELISA for herpes virus antibody uses a wallaby herpes virus antigen and it may not detect antibody to all possum herpes viruses. In order to circumvent this possible problem, reactivation of latent virus in sero-negative immunocompromised possums was attempted. Tissues were tested for cytopathic effect of virus on sensitive possum cell lines. By using this strategy it may be possible to isolate not only a herpes virus but also other possum viruses as well. Twenty possums were trapped in the Paraparaumu area. They received corticosteroid (Dexfort 0.3mg/kg) every week for five consecutive weeks. Nasal and conjunctival swabs were taken on the day of the fifth administration of Dexfort and the day after. Animals were sacrificed one week after the last Dexfort treatment and various tissues were collected. These included trigeminal and dorsal root ganglia (TG and DRG), lung, small intestine, liver, spleen and kidney, and also faecal samples. TG, DRG, lung and small intestine were explanted onto three different cell cultures, an opossum kidney (OK) cell line, a possum kidney (PK) cell line and a potorous kidney (PTK) cell line. A 10 percent tissue homogenate of lung and small intestine was also cultured on a monolayer of OK, PK and PTK cells. No effect of a virus has been detected from these tissues after three passages. Further experiments are in progress to attempt to isolate virus from liver, spleen and faecal samples.

  • Establishing and characterising a panel of possum primary cell cultures;

The success of primary isolation of viruses is largely dependent on the use of cell lines for this isolation. The greater the number of different types of cell lines that are used then the higher the chance is of successful virus isolation. Macropodid Herpes virus 1 (MaHV-1) grows much better in marsupial cells than in eutherian cells. In order to increase the chances of success in the isolation of possum unique viruses, three possum primary cell cultures have been established. They were derived from trigeminal ganglia, dorsal root ganglia and kidney cortex of the possums. Before these primary cell cultures could be used for virus isolation, they required further characterisation.

In order to monitor the cultures during serial passage, calculate cell yields and the dilution factor required at subculture, cell growth curves of these primary cell cultures have been determined. Trypsinised cell suspensions of each primary culture were diluted to 105 cells/ mL, 3.3 × 104 cells/ mL, and 104 cells/ mL. Three 24-well plates were seeded, one at each cell concentration, with 1 mL per well. The plates were incubated in a CO2 incubator. Cells in three wells of each plate were trypsinised and counted at a 24-hour interval for 7 days. The population doubling time of each primary culture was obtained from the log phase of the cell growth curve (Table 1).

The ability of these primary cultures to support the growth of virus has been determined. MaHV-1 was used to test the susceptibility of these primary cultures to infection by a marsupial herpes virus. A MaHV-1 viral stock was titrated out on the primary cultures. An opossum kidney (OK) continuous cell line was used as a control. The titre of the viral stock was 3.50 × 107 TCID50/ mL when titrated on OK cells. The titres were 5.36 × 105 to 7.96 × 107 TCID50/ mL when titrated on the primary cultures. All the new cell lines supported the growth of MaHV-1 and some gave higher titre values than lines currently available suggesting that they are more sensitive. Hence, these new cell lines should enhance our ability to isolate viruses which are unique to possums.

  • Investigating the susceptibility of possum trigeminal and dorsal root ganglia primary cell cultures to the infection of MaHV-1.

No major outbreaks of disease from possum colonies were reported over the past year. Tissues samples were obtained for virus culture from three possums, which had been scouring. No viruses were cultured from these samples.

  • Investigating the outbreaks of disease in possums that may be of viral origin with the aim of isolating possum viruses.

Wallaby herpes viruses. At least two types of herpes viruses have been isolated from wallabies and kangaroos. Only MaHV-1 has been tested in possums. It would be extremely useful if MaHV-2 or other viruses could be isolated from wallabies present in New Zealand as it is difficult to import such biologicals. The virus might be useful for possum biocontrol but could also be used to screen for antibodies against MaHV-2 and its related herpes virus in possums. At least six species of wallabies have been introduced into New Zealand (Warburton, 1986). They are pama wallaby, dama wallaby, black-striped wallaby (Macropus dorsalis), swamp wallaby (wallabia bicolour), brush-tailed rock wallaby (petrogale penicilata) and Bennett's wallaby (Macropus rufogriseus rufogriseus). In 1978 12 out of 23 sera of parma wallabies from Kawau Island, New Zealand had neutralising antibodies against MaHV-1. One of the sera was positive at 1:128 (Webber & Whalley 1978). Investigations designed to isolate viruses from wallabies have recently started at AgResearch Wallaceville. Eighteen parma and 36 dama wallabies from Kawau Island were trapped and blood samples were obtained. Serum neutralising tests against MaHV-1 were carried out on these sera and all sera collected so far have proven to be negative. Another sample of wallabies from Kawau Island will be tested in the near future.

5.11 PBV 271

Programme Title: Transgenic worms for the control of possums
Programme Leader: Dr Chuck Shoemaker
Institution: AgResearch

Summary

  • Complete the draft of a paper for submission to an international journal, and distribute it to referees for comment.
  • Perform approximately weekly experiments designed to obtain successful expression of marker genes in Pt, using C. elegans and Pt promoters, through rational method modifications of microinjections and microprobe technology.
  • Perform approximately monthly experiments designed to obtain successful expression of marker genes in Pt, using pantropic virus injection and/or infection, through rational method modifications of microinjection technology.

Background

The goals of this project were to develop the technology to enable the possum parasite Parastronglyloides trichosuri (Pt) to be used as a vector for genes expressing proteins which could interfere with reproductive success, growth or longevity of it's host

Approach & Outcomes

Milestone 1:

We have not written a manuscript for submission to an international refereed journal but have completed a report of the main findings of our work towards transformation of P. trichosuri. This report has been submitted to the NSSC for publication as part of the proceedings of the recently held workshop on possum biocontrol and will be expanded to a manuscript. Additional data supporting the conclusion that we have achieved transformation has been gathered in recent months and will be included in the manuscript (see below).

The conversion of the NSSC proceedings into a full manuscript has been hampered by the resignation (at short notice) of Dr Steven Skinner.

Milestone 2:

We performed approximately weekly microinjection experiments until April 2001, when it was necessary to pause due to the NSSC Possum Biocontrol meeting held in early April and the departure of Dr Steve Skinner in May 2001. Weekly microinjection of parasitic females has now resumed under Dr Warwick Grant, utilizing new vectors as described below. The key developments during the period under review have been:

Transformation utilising the P. trichosuri hsp-70 promoter:

Transformation of P. trichosuri has been achieved by microinjection of parasitic adult females with a construct in which the expression of bacterial _-galactosidase and green fluorescent protein (GFP) was under the control of a promoter derived from a P. trichosuri gene with homology to hsp-70. This construct gave strong expression in subset of what appear to be gut cells of C. elegans when transformants were stained for _-galactosidase activity. This staining pattern is very similar to the apparent localisation of expression of the likely homologue of this gene in C. elegans (Heschl & Baillie, 1990).

Similar but much weaker expression was seen in transformed P. trichosuri. In this case, the transformation was carried out by microinjection of adult parasitic females with the same hsp-70 construct. The injected females were maintained in insect tissue culture medium overnight, during which time they laid a variable number of eggs. Most worms survived the injection and overnight maintenance but did not produce eggs for more than 18-24 hours post injection. The eggs and hatching larvae were transferred from tissue culture medium to NGM agar plates spread with bacteria (either an undefined faecal culture or E. coli strain HB101) and a culture of free-living worms established.

Transformation as assessed by reporter gene activity was not readily reproducible: it has been obtained on at least two separate occasions, although on both occasions the _-galactosidase staining was weak and the GFP expression was not detectable. Thus it is clear that transformation of P. trichosuri is possible but that expression from the hsp-70 promoter is weak. Substitution of an enhanced GFP reporter using the same hsp-70 promoter did not improve the sensitivity of GFP reporter function. Attempts to induce higher levels of activity by heat-shock were also unsuccessful. This is perhaps not surprising, given that the apparent C. elegans homologue of this gene is not heat inducible.

The apparently weak activity of the hsp-70 promoter left open the possibility that the transformation efficiency following injection of parasitic females may be higher than it appears when reporter gene activity is used as the criterion for successful transformation. In this case, the low transformation efficiency and the difficulty experienced in reproducing the transformation may have been due to the poor expression of the reporter gene leading to underestimation of the transformation frequency. An alternative to reporter gene activity is to assay directly for the presence of the transforming DNA by PCR of sequences found only in the construct.

Evaluation of transformation efficiency by PCR of single worms obtained from free-living cultures 2-3 generations post-transformation is show that the frequency of transformation in this experiment was very much greater than had been seen with reporter gene activity, where <1 percent of worms appeared positive. The precise transformation frequency cannot be calculated from these data because the worms serving as template for the PCR were 3-4 generations removed from the parasitic adults that had been injected. However, it is clear that a substantial fraction of the population have inherited the DNA injected into the parasitic females from whom the free-living cultures were established. Further experiments in which worms from the first and second free-living generations are assayed separately are in progress. These experiments will permit quantitation of the rate of transformation (in the first generation) and the heritability of the transformation (in the second generation).

Data collected support two additional conclusions concerning transformation of P. trichosuri. First, since the worms that were assayed were several generations after the parasitic females that were injected, it is clear that the transforming DNA is inherited and expressed for at least several generations in the absence of any selection. This suggests that transformation is stable, when it occurs. Second, these data imply that the hsp-70 promoter is weak. An alternative explanation is that the promoter is silenced in some way in the transgene. In either case, it is clear that the search for stronger promoters with activity in transgenes should continue. Ideally, the sensitivity of reporter expression and PCR should be similar.

Alternative promoters:

Two promoters that are conserved between disparate species of nematodes and have high levels of activity in C. elegans are the GATA promoters for the gut specific protease and esterase enzymes (Britton et al., 1998) and the promoter for glyceraldehyde-3-phosphate dehydrogenase (G3PDH, Qin et al., 1998).

We have obtained two constructs containing the GATA promoter driving _-galactosidase expression (p98GATA and p212GATA) and have confirmed that both constructs are strongly expressed in the gutcells of transformed C. elegans (data not shown). As expected, the _-galactosidase staining pattern is almost identical to that observed with hsp-70 expression. One preliminary transformation experiment utilizing p212GATA in P. trichosuri did not produce first generation free-living progeny with gut _-galactosidase activity. Analysis of first and second generation progeny by PCR is in progress.

We could not obtain the published G3PDH promoter construct but have initiated experiments to create one ourselves. We have obtained a 5' partial cDNA sequence of the P. trichosuri G3PDH gene from collaborators in the USA and cloning of the genomic copy of the gene plus the promoter region is in progress.

We have also obtained partial cDNA sequence of a cDNA that is highly conserved and expressed in other parasitic nematodes. It encodes an abundant secretory antigen of unknown function called either "24kDa antigen" or "p22 upper" in filarial nematodes. Cloning of the gene and associated promoter region for the P. trichosuri homologue is in progress, with the aim of creating a translational fusion to drive secretion of the reporter gene in transgenic P. trichosuri.

Milestone 3:

We have not pursued transformation using pantropic retrovirus further. This is because of the promising results utilising microinjection of adult female parasites and the publication of papers describing nematode transformation by biolistic particle bombardment (Praitis et al., 2001). We have applied for and are awaiting ERMA approval to trial particle bombardment with P. trichosuri.

Future Directions:

We are now confident that it is possible to transform P. trichosuri. This is a major milestone and the first demonstration of stable, heritable transformation of an animal parasite. The next steps in the development of this organism into a disseminating biological control agent are:

  1. To refine the transformation technology to make it more routine
  2. To define more active promoter sequences to drive transgene expression
  3. To define promoters and secretory leader sequences that drive high level secretion of transgene products and
  4. To test the ability of transgenic nematodes to survive and express transgenes in vivo in possums

Effort to refine the transformation technique will focus on improving the yield of progeny from the injected parasitic adults and on attempting to adapt the recently described biolistic bombardment technology. Biolistic techniques are an attractive alternative to microinjection because they offer the possibility of transforming large numbers of worms in a single experiment and may be applicable to free-living adults rather than parasitic adults. The combination of these factors should increase the scale of the transformation experiments that are possible and the numbers of progeny from transformed worms, thus increasing the probability of success.

We have already made some progress towards defining more active and secretory promoters (see above). These will be developed in tandem with the refinement of the transformation techniques, since it is clear that poor transgene expression is a likely component of the current difficulty in reproducing the reporter gene expression illustrated in Figures 1 and 2.

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