2002-2003 Research Results - 5.Possum Biocontrol Category

5. Possum Biocontrol Category

Blocking embryonic development in Brushtail Possums

Possum viruses

Oocyte growth factors

Approach & Outcomes

Controlled oestrus

Oral bioactives

Transgenic worms

Possum Immunology

Field trial to determine the efficacy of an oral bait BCG for wild possums.

5. Possum Biocontrol Category

This is the final year of MAF being responsible for and the reporting all the Possum Biocontrol Research programmes. (Except for the Bovine Tb programme.)

5.1 PBC 251

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

SUMMARY

Goal: Three highly possum-specific targets already cloned, will be tested to determine their immune response and their effects on embryonic development in the possum. Alternative systems of protein expression will be examined to improve protein expression.

Rationale: Unlike other aspects of reproduction, possum embryonic development has many unique developmental features that make suitable targets for controlling reproduction. Our previous projects have examined three of these:

The polarised oocyte molecules are responsible for lineage allocation into pluriblast (embryonic) and trophoblast (placental) tissues (Frankenberg and Selwood, 1998, 2001). Ten vesicle-associated molecules (VAMs, of these 9 are vesicle associated proteins [VAPs] and one is hyaluronate) were identified. Four are worth testing because of possum specificity and/or embryo lethality effects (Vap5, Vap7, vasa protein, and fetoprotein). This project examines VAP5.
The outer egg coats (CPs) are essential for normal embryonic development (Selwood, 2000) and for maintenance of blastocyst epithelia in the possum (Casey and Selwood, 2003). We have isolated 7 shell coat proteins yielding 5 amino acid sequences. Three unique proteins (CP2, CP4 and CP5) are suitable for further studies. CP4 is tested here.
Leukaemia inhibitory factor (LIF) is present in the possum at the time of implantation (Cui and Selwood, 2000) and is essential for implantation (Stewart et al, 1992). Here we examine possum immune response to LIF and the effect of LIF antibody to the target proteins.

In each group, genes have been cloned and recombinant protein expressed and purified for testing in possums. We expect that these target molecules will be possum specific.

Context of the project: To identify proteins which are essential for embryonic development and use these for immunocontraception of the brushtail possum. In our previous projects we established that a number of molecules, many unique to marsupials, are essential for embryonic development in the brushtail possum. The substances examined span all of pre-implantation embryonic development; VAPs are essential for early development and cleavage, CPs are essential at blastocysts stages and LIF is essential at implantation and for proliferation of germ cells.

Objective 1 Title: Assessment of vesicle-association protein, VAP5.

Objective Leaders: Professor Lynne Selwood, Dr Shuliang Cui

Objective 1: Aims

The gene vap5 has been already cloned and a recombinant protein expressed. It is a protein that was isolated from vesicle-rich oocyte fractions, which showed possum unique sequences. We will determine whetherVAP5 recombinant protein initiates an immune response in the possum and begin assessment of its effect on development and oogenesis in in vitro assays. Alternative protein expression protocols will be tested because of the instability of this protein.

OBJECTIVE 1: CONTEXT

The possum oocyte is markedly polarised, with pronuclei and mitochondrial-rich cytoplasm at one pole and vesicle-rich cytoplasm at the other (Frankenberg and Selwood, 2001). The polarised discharge of the vesicle-rich cytoplasm acts to polarise the conceptus and leads to separation of the first two lineages, the pluriblast that forms the embryo and the trophoblast that forms the placenta (Frankenberg and Selwood, 1998). By running differential PAGE of the mitochondrial-rich and vesicle-rich cytoplasm we have been able to identify 7 vesicle-associated molecules (VAMs) of which three (vesicle associated proteins [VAPs] VAP5, 7 and fetaprotein) have been selected for further study and a protein associated with the germ cell lineage markers, VASA protein (Tanaka et al, 2000), that is located in the mitochondrial-rich fraction (Frankenberg and Selwood, 1998). These molecules are important for early development and for development of the germ cells, sperm and eggs.

OBJECTIVE 1: APPROACH

Using the amino acid sequences previously determined from differential PAGE of oocytes we have identified two vesicle-associated proteins VAP 5 and VAP 7 and a third Vasa protein from our EM studies. VAP5, a 35 kDa protein is examined here. Because many of the proteins are held in separate vesicles and assembled just before release in the possum (Frankenberg and Selwood, 1998) we suggested that they may be unstable or difficult to store.

Protein Expression:

Initially the GST (glutathionione S-transferase) protein expression system was used to produce VAP5 protein but purification of the protein proved difficult and it was unstable once purified. Initial antibodies and immune response trials were begun using the fusion protein (GST-VAP5).

Alternative expression systems tested for VAP5 protein expression.

Two alternative systems were examined with respect to VAP5 protein production.

pMAL-c2 system. One was the pMAL-c2 system, in which VAP5 was fused with maltose-binding protein (MBP), which binds to maltose to provide a method of purification. The modified open reading frame of the vap5 cDNA was transferred to pMAL-c2 in the BamHI site, which was further used to transform the bacterial cells of JM109 strains. The fusion protein was also induced with isopropyl-b-D-galactosidase by culturing the cells at 37^oC and released from its cytoplasm by sonication. MBP-VAP5 binds to maltose while others are eliminated by washes. The fusion protein can be eluted or the VAP5 is separated by the protease Factor Xa. VAP 5 protein production works well when the protein is produced in small quantities.

Gateway Cloning Technology System The system developed for CP4 will now be applied to VAP5 (see Objective 2, Approach)

Synthetic VAP5 peptides. Because of the initial difficulties with the VAP5 protein expression, we also commissioned the preparation of 20 amino acids as a synthetic VAP5 peptide (Auspep. Pty Ltd.). The peptides were selected on the basis of number (>15) and sequences most likely to be antigenic. The cDNA sequence of vap5 was analysed and converted to its coded amino acid sequence, and the polypeptide sequence was analysed for its physio-chemical properties by computer programs and databases on the Internet. Three motifs along the polypeptide were identified as highly antigenic regions that have solvent accessibility. These motifs are candidate epitopes to initiate immune response in animals. The motif with highest antigenicity has been synthesized.

Subsequently, the peptides were used either directly or after conjugation with keyhole limpet haemocyanin (KLH) as a carrier to immunise mice to raise polyclonal antibodies.

Immune trials in possums

Three possums were trapped, housed in a captive colony and a test bleed done to determine basal levels of response. Possums received 300µg VAP5 recombinant protein (pMAL fusion system) in Freund's complete adjuvant (CFA) in sterile saline. Subsequently, possums received VAP5 recombinant protein in Freund's incomplete adjuvant (IFA) for up to three boosts. Controls received saline and adjuvant test bleeds were performed 1 week after boosts. One possum received the GST-VAP5 protein. Possum responses were tested with ELISA using Possum IGg kindly provided by Dr. B. Buddle (Wallaceville). ELISA tests were run at dilution of 1/200-1/52,100.

Assessment of VAP5 antibody on oogenesis

Histological assessment of VAP5 distribution were made in normal follicles by immunostaining with VAP5 polyclonal antibody raised in mice.

In vitro assay of the effect of VAP5 Antibody. Follicle cultures (n=10 cultures, with 10-20 follicles/culture) were established in the presence of VAP5 polyclonal antibody and the effect on follicle and oocyte development determined and compared to controls. Primordial and early primary follicles dissected from the ovary were cultured as small clumps (10-20 per clump) in DMEM + 3% FCS, containing insulin, tranferrin and selenium at 1% (V/V), BSA 5mg/mL and 1.0 IU/mL follicle stimulating hormone (FSH) in 25 L microdrops under equilibrated oil for 48 hours. In experiments, follicles were exposed to the heat inactivated mouse polyclonal antibody serum at 0.1 and 0.01 concentrations and controls were exposed to control serum at similar concentrations. After culture and assessment the clumps of follicles were fixed in 4% paraformaldehyde for 24hrs, double embedded in agar and paraplast, then sectioned at 8m and stained in haematoxylin and eosin.

Because we cloned vap5 using ovarian tissue (Cui and Selwood, in preparation) we expect it to be derived from the oocyte, follicle cells or stroma. As the degenerate probe was originally derived from oocyte tissue it is most likely to be oocyte specific, but possibly originally derived from the follicle cells.

OBJECTIVE 1, OUTCOMES:

VAP5 protein production

GST-VAP5: The plasmid construct pGEX2T-VAP5 with correct orientation was transformed into and the bacterial cells expressed an inducible 35 kDa protein. The protein expressed proved difficult to separate from the fused glutathione S-transferase (GST) and the fusion protein was used in early immune trails for this reason.

MBP-VAP5: This system is a better expression system for VAP5 protein but the large-scale preparation needs to be improved. The MBP-VAP5 fusion protein produced in this system was used successfully to immunize animals and has shown some positive responses. Western blot analysis showed that the antiserun against VAP5 collected from the immunized possum was binding not only to the MBP-VAP5 but also the GST-VAP5, suggesting that the response was due to VAP5.

Synthetic VAP5 peptides

The synthetic peptides were produced by Auspep but did not raise a satisfactory polyclonal antibody in mice as demonstrated by Western blot or by ELISA. The synthetic peptide has now been conjugated to KLH and the immune protocol commenced to raise polyclonal antibodies in mice before proceeding to possums.

VAP5 possum immune response

The immune response was tested with ELISA in dilutions of 1/200 to 1/52,100. Controls (saline) were at the same level as the initial bleeds. Levels rose progressively over the 3 months of the immunization protocol, but were quite low initially. The titre was 1 in 51,200 or greater. GST-VAP5 titre level was 1/25,600 at the final bleed.

Therefore despite our reservation about the stability of the protein an immune response was raised and eventually reached high levels.

The Protein preparation is in progress to deliver VAP5 in the 2003 breeding season, after testing the Gateway system.

Assessment of VAP5 antibody

Normal standards for oogenesis (Eckery et al., 1996; Frankenberg et al., 1996; Frankenberg and Selwood, 2001) and for early embryonic development (Frankenberg and Selwood ,1998; Frankenberg et al ,2001; Casey and Selwood, 2003) for the possum and for the dunnart have already been developed.

Immunostaining

The polyclonal mouse antibody raised against VAP5 was not suitable for staining paraformaldehyde fixed, paraffin embedded sections, but was active in in vitro assays.

In vitro assay to test the effect of VAPs on oogenesis.

Follicles were cultured in clumps for 48 hrs at the primordial and early primary stages in the presence or absence of VAP5 polyclonal antibody at concentrations of 0.1 and 0.01 and compared to controls using pre-bleed sera. Because of the time of the year, dunnart follicles were used for this assay. A proportion of follicles progressed to the secondary follicle stage and a small proportion to antral stage follicles.

An interesting and important result was the concentration dependent deleterious effect of the antibody on oocytes in primary follicles at the stage in which vesicle deposition occurs and oocyte growth is initiated. As a result oocytes in primary follicles, expanding late primary follicles and possibly secondary follicles at low antibody concentrations were affected (Table 1). These oocyte were frequently shrunken and had fewer vesicles than controls. They looked darker and less transparent before fixation. The timing of action appeared to be coincident with the first appearance of vesicles in the oocyte. Early gene expression of VAP 5 in the ovary (Cui and Selwood, in prep.) occurs at this time.

Table 1. The effect of VAP5 polyclonal antibody on oocyte development in follicles in vitro

Percentage of oocytes that failed in vitro

Oocytes in follicle type	0.1 Ab, expt.	0.1, control	0.01 Ab, expt.	0.01 control
Primordial	17	-	38	0
Primary	66	0	73	14
Late Primary	100	0	82	8
Secondary	21	20	62	41
Antral	0	0	-	-

OBJECTIVE 2 TITLE: ASSESSMENT AND CLONING OF COAT PROTEINS

OBJECTIVE LEADERS: Professor Lynne Selwood and Dr. Shuliang Cui

Objective 1: AIMS

The coat protein (CP4) that has already been cloned, characterized and expressed as a recombinant protein is a novel protein (Cui and Selwood, 2003) and has been reported on previously. We will test its ability to raise an immune response and further examine the effect of its antibody on uterine tissues in vitro. We also aim to test alternative protein expression protocols to facilitate production.

Objective 2: CONTEXT

Because the shell coat is marsupial specific and we have demonstrated earlier that in possums it is essential for normal blastocyst formation and for maintenance of blastocyst epithelia (Selwood, 2000; Casey et al, 2002; Casey and Selwood, 2003), we expect an immune response to the shell coat proteins to terminate embryonic development well before implantation stages. CP4 is the first of the possum coat protein genes to be cloned and expressed as a protein and as expected has been shown to be a novel protein (Cui and Selwood, 2003). Briefly, the first half of the molecule has homology to crystallin (turtle lens protein) and enolase and the last half is the unique segment. Because of the connection to crystallin and the characteristics of the shell coat we think it possible that CP4 is a structural protein that confers on the coat its stability, transparency and tensile nature and possibly its absorbent qualities.

Objective 2: APPROACH

Protein production - CP4

The major problem with cp4 expression is the low yield and the instability of the protein once expressed using the gst system. This may not matter if the recombinant protein elicits a satisfactory immune response in possums. The other method tested was the gateway cloning technology system which is universal system allowing a rapid and highly efficient route to protein expression and functional analysis.

The first step towards the Gateway expression of recombinant proteins was to obtain the attB substrate, i.e the DNA fragment of the gene of interest with the recombination site. cp4 cDNA was amplified using a pair of specific primers, which is a lambda phage based site-specific recombination. The second step was to generate an entry clone. The DNA recombinantion sequences (attL, attR, attB and attP) and Clonase enzyme mix were used for transferring the gene into the entry vector. We have successfully created an entry clone for cp4 gene using the pDONR 221 vector, which is the donor of the sequence for further cloning of the gene of interest into the destination vectors. Using the LR Clonase, we have created two expression constructs using pDEST 15 and pDEST 17. The third step was to induce the expression of the recombinant proteins so that the protein can be over-expressed with E. coli cells. Using the E. coli cells strain BL 21 (Promega), 0.4 mM IPTG was able to induce GST-CP4 fusion protein with the pDEST 15 vector and (His)₆-CP4 fusion protein with the pDEST 17 vector. The over-expressed fusion proteins were estimated to be about 40 % of the total protein. The fourth step was to release the recombinant protein from the cytoplasm and make them soluble in non-denaturing conditions. The cells were cultured at 37^oC until the cultures were in the mid log phase (about 1.5 hour) by vigorous agitation. Cells were harvested by centrifugation at 3000 x g for 15 minutes at 4^oC. Various protocols were tried to lyse the cells and solubilize the fusion protein, and found that the CP4 protein was not soluble in most of the suggested buffers. The proteins were solubilized with non-ionic detergent at different stages of the preparation procedures. The final step was to purify the fusion protein by affinity chromatography. We use the glutathione sepharose 4B beads (Amersham Biosciences) to purify GST-CP4 and the ProBond (Invitrogen) to purified His-CP4.

Testing possum immune response:

Three possums were trapped, housed in a captive colony and a test bleed done to determine basal levels of response. Possums received 300µg CP4 recombinant protein in Freund's complete adjuvant (CFA) in sterile saline. Subsequently, possums received CP4 recombinant protein in Freund's incomplete adjuvant (IFA) for up to three boosts. This recombinant protein was used still bound to GST because of problems with protein stability once purified. Controls received saline and adjuvant. Test bleeds were performed 1 week after boosts.

In vitro assays

In vitro assays using uteri obtained at the late follicular phase when uteri are about to enter the proliferative phase (Casey et al, 2002) were used to determine the level of CP4 expression in vitro and whether this can be up regulated in vitro using progesterone or oestradiol. This would make it more feasible to examine the protein in the medium. The uteri were cultured as slices and as monolayers.

Slice cultures 1-2mm thick were cultured in DMEM containing progesterone and oestradiol at physiological and 10x physiological levels for 18 hours. The incubation media were collected and frozen and the slices, after washing in PBS, were snap frozen for later analysis.

Monolayer cultures were established over Matigel, a basement membrane matrix, in DMEM in 96 well plates after cell dissociation using 0.25% collagenase. Cultures were maintained for 7-14 days to confluence and glandular formation. The effect of the antibody on uterine glandular structures were assessed in addition to analysis of the proliferation in response to hormones and CP4 antibody.

Objective 2: OUTCOMES

Protein production - CP4

It was well demonstrated that the GST-CP4 and the His-CP4 were produced in an inducible manner, which gives higher yield than the pGEX2T system. The recombinant protein purification has been achieved for GST-CP4 and that for His-CP4 is in progress. Alternatively, the protein can be easily produced with other cell types rather than bacteria.

Possum immune response

The immune response was tested with ELISA at dilution of 1/200 to 1/52,100. The controls (saline and GST) were negative and both possums had an immune response to CP4. One had a low response, possibly due to degradation of the protein with storage, but the other had high levels of response. The titre level was 1 in 52,100. Levels rose progressively over the 3 months of the immunization protocol and for a further 3 months. Therefore despite our reservation about the stability of the protein an immune response was raised but the level was only reasonably high in one possum. Saline and adjuvant controls were negative as was the GST run in the ELISA tests.

The Protein preparation is in progress to deliver CP4 in the 2003 breeding season, using the modified Gateway Technology System.

Begin assessment of effect of CP4 in vitro

Possum coat protein CP4 production is being tested against uterine monolayers on matrigel, in which glandular formation occurs in vitro. Monolayers are derived most successfully on the day of ovulation (day 0) and for 2-3 days thereafter reflecting the high level of mitoses encountered in uteri at the uterine proliferative stages. The glandular epithelium and lumen stain positively with labelled antibody and we expect, based on earlier trials, that production or secretion of CP4 will be inhibited by the presence of antibody in the media. This is still to be confirmed. Media analysis in still being defined and histological analysis is in progress. Media analysis using Coomassie blue staining of media is unsatisfactory and a silver staining technique is being refined. In addition application of the Northern analysis developed for uterine tissue to demonstrate expression patterns in vivo (Cui and Selwood, 2003) is in progress. Cell proliferation in matrigel monolayers can be significantly upregulated in the presence of low progesterone (5 and 11 ng/mL) and high oestradiol (23, 140 and 230 pg/mL). Assessment of CP4 production in these various assays is being developed.

Immunostaining

The polyclonal mouse antibody raised against CP4 stains individual cells in uterine glandular epithelium but also has considerable levels of background stain. We are revising the concentration of Ab used in addition to blocking reagents to eliminate the background. However, because the first part of the CP4 molecule is homologous to crystallin, which in turn has homology to enolase, we expect it to stain a variety of other tissues especially those with high energy turnover where enolase would be encountered. Because the unique section of CP4 lies in the last half of the molecule, future antibody production would be based on the second half of the molecule to increase specificity.

In vitro assay of the effect of CP4 Antibody

CP4 antibody in concentration of 10 L/mL causes significantly higher proliferation of uterine cells but not at 1 L/mL. The biological significance of this is unknown at present. It is possible that uterine proliferation which is down regulated by rising progesterone levels during early pregnancy might also be down regulated by CP4, which is present at high levels then. The antibody might block the down regulation, causing interruption to normal uterine endometrial cycling.

Objective 3 TITLE: ASSESSMENT AND CLONING OF IMPLANTATION PROTEINS

Objective 3: Research Leaders: Dr. S. Cui and Professor L. Selwood.

Objective 3: AIMS

On the basis of what is known of LIF biology, interfering with normal LIF function either through LIF or its receptor, should affect normal embryonic development at implantation stages (flat embryo) and also primordial germ cell (PGC) proliferation between the primitive streak stages to soon after birth (Eckery et al, 1996, Frankenberg et al, 1996). Using the possum LIF gene and recombinant protein already produced we will examine whether LIF protein induces an immune response in reproducing possums and begin assessment of its effect on embryonic development and gametogenesis. We know that it enhances proliferation of PGC in vitro, so we will test the effect of LIF antibody on PGC proliferation in vitro. We aim to test alternative protein expression protocols to facilitate production.

Objective 3: CONTEXT:

LIF protein is essential for implantation (Stewart et al, 1992) and is also one of the growth factors required for proliferation of the germ cells (Matsui et al., 1992). We have previously cloned and characterised the possum LIF gene (pLIF) and found expression patterns similar to eutherian mammals (Cui and Selwood, 2000).

Objective 3 APPROACH

Testing possum immune response:

Four possums were trapped, housed in a captive colony and a test bleed done to determine basal levels of response. Possums received 300-µg LIF recombinant protein in Freund's complete adjuvant (CFA) in sterile saline. Subsequently, possums received LIF recombinant protein in Freund's incomplete adjuvant (IFA) for up to three boosts. Controls receive saline and adjuvant. Test bleeds were performed 1 week after boosts. No tests were done in the breeding season.

Begin assessment of effect of LIF on reproduction

In vivo: Using the antibody to LIF, uteri from cycling and pregnant females were examined by immunocytochemistry, to determine the distribution of LIF.

In vitro: Two in vitro assays have been developed; one uses primordial germ cells (PGCs) obtained from gastrula stage embryos and from germinal ridges of PY and the other uses fibroblast feeder layers as a standard, year round LIF assay kit. For this project emphasis will be on PGC in vitro assays exposed to LIF antibody because we have already shown last year that LIF increases proliferation of PGC over 10 days. For the PGC proliferation assays, PGCs collected from gastrula stage embryos or from early PY will be cultured in microdrops (150 per 250L drop) and the effect of possum LIF antibody at 1 L/mL or 10 L/mL on proliferation assessed. PGCs from both dunnarts and possums can be used to enable more comprehensive testing throughout the year.

LIF protein production.

The LIF expression system is satisfactory but slow. We concentrated on applying the Gateway Technology to CP4, then VAP5 and if time permitted we planned to also apply it to LIF but we have not progressed to applying Gateway to LIF production.

Objective 3: OUTCOMES

Possum immune response to LIF.

The immune response was tested with ELISA run at dilution of 1/200 to 1/52,100 and showed high levels in all possums. The titre was 1/26,500. Levels rose progressively over the 3 months of the immunization protocol. Either control, saline or IFA, did not induce an immune response as demonstrated by ELISA.

Protein preparation is in progress to deliver LIF to cycling females in the 2003 breeding season, using the GST fusion protein.

Begin assessment of effect of LIF on reproduction.

In vitro: In the presence of LIF polyclonal antibody at either concentration PGC cell numbers decline markedly in vitro after day 4 of culture and rapidly fall towards control levels. (N=6, with a sample size of 150 PGC per assay). This means that in vivo LIF antibodies could be expected to inhibit PGC production and in the long-term, fertility of adults, by decreasing the number of gametes. While we haven't yet tested the sex of PGCs in these trials, the results suggest that LIF could affect both male and female fertility.

LIF polyclonal antibody in concentration of 1 L/mL causes significant increase in total cell numbers of uterine epithelial cells in matrigel monolayers after 4 days of incubation. When included with our other results this result suggests that LIF may act to limit the proliferation of uterine epithelia at early cleavage stages following the hormonal stimulation that occurs at oestrus. If this is correct, then immunisation with LIF might also act to interfere with the normal uterine cycling associated with pregnancy.

In vivo: LIF antibody was used to stain uteri at various stages of development. Positive results were encountered in the uterine luminal epithelia of possums at the 2-cell stage and at the flat embryo early implantation stage. Results were negative in uteri from unilaminar blastocyst and fetal stages. The location and timing of the staining suggests that LIF in the possum as in the mouse is involved in implantation and probably plays an essential role in implantation, even though in the possum, implantation is extremely superficial. The significance of the positive staining at the 2-cell stage is unknown, although the mouse also has a brief window of expression of LIF around the time of ovulation.

Protein expression is in progress to provide recombinant LIF for trials on male and cycling female and possums.

Additional studies on manipulation of possum husbandry to extend the breeding season and increase number of py per annum

AIM:

To extend the breeding season of female possums and increase number of PY per annum.

CONTEXT:

In Australia, as in NZ, possums have a restricted breeding season. In our colony in Melbourne, 1996-2000 it began in late March or early April and extended to September. Males have sperm all year and are potentially capable of siring young in any month. Crawford et al (1998) showed that in possum housed in colonies, exposure to males synchronized the return to ovulation after removal of pouch young (RPY). In the light of this information I decided to try to extend the breeding season of the female possums by changing the sex ratio of females to males in the colony.

APPROACH

Between 1996-2000, my colony possums followed the breeding season normal to Melbourne (Table 1). The maximum colony size was 15. Cycles were relatively synchronized. PY were removed within 7 days post partum. In 2001-2003 the female to male ration was changed to 2F or 3F to 1M. In 2001- 2003, the female to male ratio has been changed to 1F:1M or 1F:2M. Colony size varied between 20 and 22.

OUTCOMES

Following changing the sex ratio, the breeding season was extended so that breeding occurred in every month except December (Table 1). The number of PY per annum was increased by 20% and the % of females with PY per cycle was also increased. While this phenomenon has not been rigorously tested, because of space restrictions in the colony, it would be desirable to test it in NZ where colony sizes and facilities are larger.

Table 1. The effect of changed female to male sex ratio on the length of the breeding season and colony productivity. PY, pouch young; pp, post partum; RPY removal of pouch young.

	1996- 2000	2001- 2003
Sex ratio, Female :Male	3:1 or 2:1	1:1 or 1:2
Breeding season	March- September	January- November
Productivity	50% conception per cycle	May- Oct, 50-70% conception per cycle Nov.- April, 15-30% conception per cycle
PY per annum for 10 females with RPY each cycle by day 7 pp.	35	42

Additional studies on the steroidogenic nature of the follicles that persist during pregancy in the possum

AIM

To determine the steroidogenic capacity of the persistent follicles of pregnancy in the possum.

CONTEXT

The possum has follicles of unknown function, which develop and enlarge during pregnancy to a large size (3.1mm) and then become atretic at late fetal stages prior to parturition (Kean et al, 1963). Kean's findings were based on over 1000 NZ possums and were confirmed in our studies on development. Because of their possible implications for fertility control I decided to investigate whether the follicles were steroidogenic by collaboration with Dr. Ullmann, University of Glasgow.

APPROACH

During our other studies, ovaries were collected and fixed in paraformaldehyde and sent to Dr. Ullmann, University of Glasgow who examined the possum sectioned ovaries and those of another marsupial by immunocytochemistry to determine the location of 3-hydroxysteroid dehydrogenase/5-4 isomerase (3 -HSD), using a rabbit polyclonal antibody. 3 -HSD is an essential enzyme in the biosynthesis of sex steroids and its presence demonstrates steroidogenic capacity. Some follicles were removed from fresh ovaries and the follicles removed and the oocytes examined in vitro.

OUTCOMES

On examination of the follicles in vitro, we found that the oocytes were apparently normal and histology confirmed the absence of atresia. Immunocytochemistry demonstrated that 3 -HSD was present in the corpus luteum, interstitial tissues, theca of Graafian follicles and the supernumerary follicles of pregnancy (Ullmann et al, 2003). These unusual follicles were not found in the other marsupial examined.

CONCLUSIONS

Each of the targeted proteins VAP5, CP4 and LIF has sufficient possum specificity, developmental significance, levels of possum immune response and effect on developmental processes in vitro to make them suitable for testing to determine their effect on possum fertility.

HIGHLIGHTS

All 3 proteins raise an immune response in possums, which increases continuously over 3 months and over 9 months in one case.
The possum breeding season can be extended from 7 to 11 months. Hence the productivity of the colonies increases and the experimental period is extended.
LIF antibody, causes a marked decline in PGC numbers in vitro suggesting that LIF could cause decrease in numbers in male and female gametes in vivo and thus reduce fertility.
VAP5 antibody causes deterioration of oocytes in primary and secondary follicles in vitro, suggesting that development of oocytes could be inhibited.

REFERENCES

Nicholas Casey and Lynne Selwood (2003). Removal of the shell coat affects maintenance of epithelia in blastocysts of the brushtail possum in vitro. Cells Tissues Organs, 173, 21-45.

Casey, N., Martinus, R. and Selwood, L. (2002). Outer egg coats of the marsupial conceptus: Secretion and protein composition. Molecular Reproduction Development . 61, 181-194.

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.

Cui, SC and Selwood, L. (2003). Cloning and expression of a novel cDNA encoding shell coat protein, cp4, from the brushtail possum (Trichosrus vulpecula). Molecular reproduction and Development. 65: 141-147.

Cui S and Selwood, L (in preparation). Cloning and expression of a vesicle-associated protein, VAP5, a polarised oocyte protein in the brushtail possum (Marsupialia).

Eckery, DC, Tisdall, DJ, Heath, DA and McNatty, KP (1996). Morphology and function of the ovary during fetal snd early neonatal life: A comparison between the sheep and the Brushtail possum (Trichosurus vulpecula). Animal Reproduction Science, 42 551-561.

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.

Frankenberg, S. and Selwood, L. (1998). An ultrastructural study of the role of an extracellular matrix during cleavage in a marsupial, the brushtail possum. Mol. Reprod. Dev. 50, 420-433.

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

Frankenberg, S., Newell, G. and Selwood, G. (1996). A light microscopic study of oogenesis in the brushtail possum, Trichosurus vulpecula. Reprod. Fertil. Devel. 8, 491-508.

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.

Kean, R.I., Marryatt, RG and Carroll A.L.K. (1964). The female urinogenital system of Trichosurus vuilpecula (Marsupialia). Aust. J. Zool. 12, 18-41

Matsui,Y, Zsebo, K, Hogan, BLM (1992). Derivation of pluripotential embryonic stem cells from mrine primordial germ cells in culture. Cell 7-: 841-847.

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

Stewart CL, Kaspar P, Brunet LJ, Bhatt, H Gadi, I Knotgen, F and SA Abbondanzo (1992). Blastocyst implantation depends on maternal expression of leukaemia inhibitory factor. Nature 359, 76-79.

Toyooka, Y, Tsunekawa N, Takahashi, Y, Matsui, Y Satah, M and Noce, T. (2000) Expression and extracellular localisation of mouse Vasa homologue protein during germ cell development. Mechanisms of development, 93, 139-149.

Tanaka, SS, Toyooka, Y, akas, R, Katoh-Fukui, Y, Nakaharra, Y, Suzuki, R, Yokoyama, M and Noce, T. (2000). The mouse homologue of Drosophila Vasa is required for the development of male germ cells. Genes and Development, 14, 841-853.

Ullmann, SL, Russell, AJ, Mason, JI and Selwood, L. (2003). Species differences in the ovarian distribution of 3-hydroxysteroid dehydrogenase/5-4 isomerase (3 -HSD) in two marsupials: the brushtail possum Trichosurus vulpecula and the grey, short-tailed opossum Monodelphis domestica. Reproduction 125, 65-73.

PUBLICATIONS

Cui, SC and Selwood, L. (2003). Cloning and expression of a novel cDNA encoding shell coat protein, cp4, from the brushtail possum (Trichosrus vulpecula). Molecular Reproduction and Development. 65: 141-147. (Attached).
Nicholas Casey and Lynne Selwood (2003). Removal of the shell coat affects maintenance of epithelia in blastocysts of the brushtail possum in vitro. Cells Tissues Organs, 173:21-45. (Provided, 28.2.03).
S. L. Ullmann, A. J. Russell, J.I. Mason and L. Selwood (2003). Species differences in the ovarian distribution of 3b-hydroxysteroid dehydrogenase /D^5_4isomerase (3bhsd) in two marsupials: the Brushtail Possum, Trichosurus vulpecula and the Grey, Short-tailed Opossum, Monodelphis domestica. Reproduction 125, 65-73. (Attached).

CONFERENCE PROCEEDINGS

S.Cui and Selwood, L. (2002). A unique protein, possum shell coat protein 4 (CP4) is a possible target for immunocontraception in the brushtail possum. Australian Mammal Society, 48^th Annual Scientific Meeting, Warrnambool, Victoria Australia.

5.2 PBC 252

Programme Title:	Possum viruses
Programme Leader:	Tao Zheng
Institution:	AgResearch CRI

Summary

To isolate viruses from possums, and assess their usefulness for the biological control of possums.

Aims:

The goal for this programme is to isolate viruses from possums, and assess their usefulness for the biological management of possums.

Determine the sensitivity of a nested polymerase chain reaction (PCR) for detection of herpesvirus polymerase in faecal samples, using macropodid herpesvirus -1 as a positive control.
Establish a RT-PCR assay for the detection of coronaviruses based on degenerated primers and determine its sensitivity in detection of coronaviruses in faecal samples, using a bovine coronavirus as a positive control.
Detect herpesvirues and coronaviruses in possum faecal samples collected from different geographical areas by PCR and RT-PCR.
Culture any PCR/RT-PCR positive possum faecal samples on cell lines for the isolation herpesvirus/coronavirus.
Determine current prevalence of anti-herpesvirus antibody in possum from Shannon by ELISA using antigens from a wallaby herpesvirus.
Culture 50 possum faecal samples collected from areas where cross-reactive antibodies to antigens of a wallaby herpesvirus have been identified previously.
Characterisation of possum enteroviruses.

Background

Introduction:

The primary goal for this programme is to isolate viruses from possums and to assess their usefulness for the biological management of possums. Virus and virus-like particles were observed in possum intestinal contents by electron microscopy (EM), including adenovirus, herpesvirus, coronavirus and coronavirus-like particles (Rice and Wilks, 1996). Herpesvirus, adenovirus and coronavirus have the potential to be developed as vectors to deliver bioactive molecules for the biological management of possums. Virus culture is a key method for possum virus isolation. It is essential that the isolated possum viruses are culturable to enable further genetic manipulations in vitro. As viruses or virus-like particles were identified from only 23/100 possum intestinal contents by EM (Rice and Wilks, 1996), to warrant the success of isolation of a possum virus, large numbers of intestinal contents need to be processed. Pre-screening possum intestinal contents by polymerase chain reaction (PCR) or reverse-transcriptase PCR (RT-PCR) targeting herpesvirus and coronavirus using consensus primers will significantly improve the efficiency for the isolation of possum viruses. In addition, intestinal contents of possums will be cultured for isolation of viruses.

Achievement of objectives:

Determine the sensitivity of a nested PCR for the detection of herpesvirus in possum faecal samples

Pre-screening intestinal samples by PCR would significantly improve the efficiency for virus culture. A nested PCR targeting the DNA polymerase of herpesviruses was established. Its sensitivity for the detection of herpesvirus in faecal samples was determined using the macropodid herpesvirus-1 (MaHV-1) as a positive control. Bulk MaHV-1 was cultured on opossum kidney (OK) cell line, and its infectivity was determined being 6 × 10⁹ plaque forming unit (pfu)/ml. A 20% (w/v) possum faecal extract was prepared. A serial of 10-fold dilutions of MaHV-1 was made in the possum faecal extract, and DNA was then purified. Nested PCR was then conducted on the purified DNA samples. The detection level initially was approximate10⁶ pfu/ml MaHV-1, when using standard PCR condition. It indicated that there were PCR inhibitors in faecal samples that co-purified with the DNA. Therefore, the PCR conditions were modified by including a few cycles of reaction with lower annealing temperature, and by adding bovine serum albumin to the reaction. The sensitivity of the test was increased by approximately a thousand fold. The modified nested PCR method was used to detect possum herpesvirus in faecal samples.

PCR and culture for isolation of viruses from possum intestinal contents

Intestinal contents were collected from 23 wild possums shot from Shannon area of North Island, where MaHV-1 cross-reactive antibody serum positive possums had been detected by ELISA from a previous study (Zheng, unpublished data). Nested PCR targeting herpesvirus was conducted on the extracts of possum intestinal contents. Positive nested PCR results were not obtained from these samples. In the light of the possibility for the existence of other possum viruses in the intestinal contents, their extracts were subjected to cell culture for isolation of viruses. A 20% (w/v) intestinal content extract was prepared and pooled extracts were cultured on Vero, MDCK, OK and possum primary kidney (PPK) cell lines. PPK cell culture was developed in this lab previously. Two suspect virus isolates that causing cytopathic effect (CPE) on PPK cells were identified from two sets of pooled intestinal contents from apparent healthy possums, while such cytopathic effect was not observed in the parallel cultures using Vero cells (African green monkey kidney cells), MDCK cells (canine kidney cells) and OK cells (opossum kidney cells). The cytopathic effect was not eliminated by filtering the isolates through a 0.2 µm filter. It was likely that the two isolates were viruses and were designated as W1 and W6. The priority of the research has been adjusted to characterise the two viral isolates since.

Establish a RT-PCR assay for the detection of coronaviruses based on degenerated primers and determine its sensitivity in detection of coronaviruses in faecal samples, using a bovine coronavirus as a positive control; detect coronaviruses in possum faecal samples collected from different geographical areas by RT-PCR; culture any RT-PCR positive possum faecal samples on cell lines for the isolation coronavirus; determine current prevalence of anti-herpesvirus antibody in possum from Shannon by ELISA using antigens from a wallaby herpesvirus; culture 50 possum faecal samples collected from areas where cross-reactive antibodies to antigens of a wallaby herpesvirus have been identified previously.

These objectives were amended during the year, as efforts were concentrated on the characterization of possum enteroviruses as approved.

Characterisation of possum enteroviruses

The two isolates, W1 and W6, were plaque purified twice. The plaque size varied and both small and large plaques from each isolate were purified. Sequences of a ~560 bp genomic fragment were identical from small and large plaques of each virus.

Electron microscopy

W1 and W6 from cell cultures were concentrated by differential centrifugation. The virus preparations were stained with 2% phosphotungstic acid (pH 7.25) and examined using Philips CM100 BIO electron microscope. Viruses were nonenveloped small round viruses, 27-30 nm in diameter with icosahedral symmetry (Figure 1).

Buoyant density of W1 and W6 in caesium chloride (CsCl)

Possum viruses W1 and W6 banded at a density of 1.35 g/cm³ in CsCl, which is in agreement with enteroviruses.

Nucleic acid type

Nucleic acid type (DNA/RNA) of isolates W1 and W6 were determined by using 5'-bromodeoxyuridine (BUdR), a DNA virus growth inhibitor. Test results showed that the growth of both W1 and W6 were not inhibited by the addition of BUdR in the culture medium, indicating both W1 and W6 are RNA viruses.

Genomic sequences

Based on morphology and nucleic acid type, W1 and W6 are probably members of the picornavirus family. RNA from cultures of W1 and W6 were purified and were amplified by RT-PCR using consensus primers derived from enteroviruses.

A piece of ~ 560 base pairs (bp) PCR products were obtained and sequenced. Pairwise comparisons were conducted between W1 and W6 and other enteroviruses. W1 and W6 share 80.2% identity, and they share 68.1-73.4%, 67.6%, 57.6% and 53.8% identity with bovine enteroviruses, a sheep enterovirus and two human enteroviruses, a coxsackievirus and a poliovirus, respectively.

These preliminary results suggested that W1 and W6 are enteroviruses, which are members of the picornavirus family.

Developing research toolbox (reagents)

Virus purification

Purification of the newly isolated possum enteroviruses is necessary for the generation of immunoreagents and to characterise and assess the viruses for biological management of possums. Two isolation protocols were established to obtain pure virus preparations. Protocol A utilised a differential centrifugation technique. Briefly, the Protocol A includes the infection of cell culture with a possum virus; flasks were frozen and thawed when cytopathic effect (CPE) was fully developed; low speed centrifugation (3000 ×g, 15 min, 4°C) to get rid of large cell debris; high speed centrifugation (18,000 ×g, 15 min, 4°C) to remove fine cellular particles and ultracentrifugation (100,000 ×g, 180 min, 4°C) to precipitate viruses. Protocol B employed polyethylene glycol (PEG) precipitation and buoyant density-gradient sedimentation techniques. Briefly, Protocol B involved the infection of cell culture with a possum virus; flasks were frozen and thawed when CPE was fully developed; low speed centrifugation to remove large cell debris; the precipitation of viruses from culture medium by the addition of PEG 8000 and high speed centrifugation (15,000 ×g, 20 min, 4°C) and finally ultracentrifugation (120,000 ×g, 14 hr, 4°C) on a performed 5-40% w/w CsCl gradient

Protocol A was a fast and economical method for possum virus purification, but the virus was still contaminated with small amount of cell components, as observed under the electron microscopy (EM). This purification method would be useful for some applications where semi-purified virus is required, such as the preparation of viral antigens for immunoassays. Viral preparations that were purified by Protocol B were very pure under EM. This preparation is useful for the further characterisation of possum viruses and for the generation of possum virus specific hyperimmune serum. The generation of rabbit anti-possum virus hyperimmune serum is progressing.

Challenge possums with possum enteroviruses challenge

Experimental design

16 possums were captured from Upper Hutt. They were serum negative against W1 and W6, determined by virus neutralisation test (VNT).

The inocula were the third passage of W1 and W6. Viruses were harvested when cells exhibited more than 90% CPE, using a cell scraper. After a brief sonication, virus cultures were centrifuged at 3,000g for 15 min at 4°C and then the supernatants were filtered through a 0.22 µm filter. These preparations were titrated and adjusted to 10^5.6 TCID₅₀/ml and used as inocula in the challenge trial. Mock-infection inoculum was prepared from PPK cells in a similar manner.

Three possums were inoculated with W1 oral (0.9 ml/possum)/nasally (0.1 ml/possum) and three possums were challenge with W1 intravenously (1 ml/possum). Similarly, six other possums were inoculated with W6, three oral/intranasally and three intraveneously (Table 1). In a similar manner, four possums were inoculated with the mock-infection inoculum, two oral/nasally and two intravenously and served as controls. Virus-inoculated and mock-inoculated possums were housed in separate enclosures.

Clinical observations

No body loss or rectal body temperature rise were observed in any of the possums. One of the mock-infected (I.V.) possums had very soft faeces on day 21 post inoculation (p.i.). Very soft faeces were also observed from virus-inoculated possums # 944, # 6545, #6553 and #943 sporadically from day 8 to 26. The passage of soft faeces lasted for 1 to 2 days in individual possums.

Virus-inoculated possums shed virus in faeces

Faecal sample extracts of all possums collected on days 0, 7, 14, 21 and 26 p.i. were cultured on PPK cells for virus detection. Virus was detected from faecal samples collected on days 7, 14, 21 and 26 p.i. from possum #6553, which was orally inoculated with W1; and from faecal samples collected on days 14, 21 and 26 p.i. from possum #950, which was inoculated with W6 intravenously. All others produced a negative result for virus detection.

Virus neutralising antibody response in virus-inoculated possums

Blood samples were collected on days 0, 7, 14, 21 p.i.and when they were killed. W1 or W6 neutralising antibodies were detected from three possums inoculated with W1 and two possum with W6 by VNT (Table 1). W1 and W6 neutralising antibodies were not detected from control possums by VNT.

Table 1. Neutralising antibody response against W1 or W6 isolates in virus-inoculated possums


Days (p.i.)	W1						W6
	I.V.			Oral/nasal			I.V.			Oral/nasal
	948¹	944	6545	945	941	6553	950	949	946	947	942	943
0	-	-	-	-	-	-	-	-	-	-	-	-
7	-	1:8	1:8	-	-	-	1:4	1:128	-	-	-	-
14	-	1:4	1:4	-	-	1:16	1:4	1:64	-	-	-	-
21	-	-	-	-	-	1:16	-	1:32	-	-	-	-
31	-						-	1:8		-
32		-	-	-					-		-
33					-	1:8						-

1: Number of the animal

Preliminary conclusions

The challenge dose of W1 and W6 in this study was a sub-optimal dose for establishing infections in all virus-inoculated possums.
Isolate W1 was re-isolated from faeces from 1 of 3 possums when challenged orally;
Isolate W6 was re-isolated from faeces of 1 of 3 possums when challenged intravenously;
Oral as well as intravenous challenge of possums with W1 elicited serum antibody responses in 3 of 6 virus-inoculated possums;
Intravenous challenge of possums with W6 elicited antibody response in 2 of 3 virus-inoculated possums;
The infection of W1 and W6 may be associated with the mild diarrhoea in a proportion of the inoculated possums.

Engineering picornaviruses as vaccine vectors -a brief literature review

Exploring the possibility of using polioviruses as vaccine vectors started more than a decade ago (Evens et al 1989). Examples of replicate-competent live picornavirus vector systems are poliovirus vaccine strains, rhinovirus and coxsackievirus (Andino et al 1994, Smith et al 1998, Halim et al 2001). Various engineering strategies have been employed for developing replication-competent chimeric viruses expressing foreign antigens, mainly by replacing viral prominent antigenic surface loop with extraneous peptides (~ 10 aa) or inserting foreign antigenic sequences (up to ~ 350 aa) at the junctions of the viral polyprotein (Evens et al 1989, Andino et al 1994, Crotty et al 2001). Inoculation of susceptible animals with replication-competent recombinant viruses resulted in the induction of humoral, mucosal and cellular immunity against the inserted foreign antigens (Mandl et al 1998, Crotty et al 1999). Poliovirus recombinants were capable of inducing effective immunity to the foreign antigens in previously vaccinated animals (Mandl et al 2001). Multiple antigenic vaccinations could be achieved by the administration of cocktail chimeric viruses (Crotty et al 2001).

Approach & Outcomes

Two possum virus isolates were isolated from intestinal contents of apparent healthy wild possums. These two isolates were determined to be enteroviruses, members of the family of the Picornavirdae. Preliminary results from a challenge trial in possums indicated that these two viruses produced minimal disease in possums. Virus was re-isolated from 2 of 12 virus-inoculated possums. Serum antibody responses were produced in 5 of 12 virus-inoculated possums. These two possum viruses have the potential to be developed as vaccine vectors for possum biological management. Further studies, such as on viruses' transmissibility in possums and host specificity, need to be investigated.

Publications

Zheng, T., Napier, A.M., O'Keefe, J.S., Buddle, B.M.,: Experimental infection of possums with macropodid herpesvirus 1. New Zealand Veterinary Journal (in press)

5.3 PBC 254

Programme Title:	Oocyte growth factors
Programme Leader:	Doug Eckery
Institution:	AgResearch CRI

Summary

To sterilise female possums by immunising them against GDF-9 and/or GDF-9B

Both GDF-9 and GDF-9B are oocyte-specific growth factors. Mutations in these growth factors or their absence can lead to infertility by blocking early follicular growth. The aim of this work is to determine the effects of immunising possums against these growth factors on ovarian function and reproductive activity.

Objective 1 - Immunisation of possums against GDF-9 and GDF-9B

The aim of this work was to determine the effects of immunisation against GDF-9 and GDF-9B on ovarian function in possums. Adult female possums were immunised against either recombinant ovine GDF-9, recombinant ovine GDF-9B or KLH (controls). Following a primary immunisation and six subsequent booster immunisations, the reproductive tracts of all animals were examined to determine the effects of immunisation. Blood samples were also collected to determine antibody titres. Immunisation against GDF-9 or GDF-9B did not have any effect on body weight, cul-de-sac weight or ovarian weight compared to controls. The determination of antibody titres and ovarian histology are currently underway. Although both GDF-9 and GDF-9B have been shown to be important for follicular growth in other species, there was no apparent effect of immunisation against the ovine proteins. This could be due to the fact that these growth factors don't play important roles in possums, or alternatively, that the possum equivalents of these proteins differ enough from the ovine proteins that immunisation against the ovine proteins was ineffective. If the latter is true, it may be possible to develop a permanent form of fertility control that is species-specific to possums. A subsequent study is planned to immunise possums against recombinant possum GDF-9 and GDF-9B.

Background

Ovarian follicular growth is a complex process involving the exchange of endocrine signals between the pituitary gland and ovary as well as close communication between the various cell types within the ovary via locally produced growth factors. The oocyte (egg) has often been viewed as a passive cell type that requires nurturing and direction from the surrounding somatic cells. More recently, mounting evidence has demonstrated that the oocyte is instead a very active cell, and that it plays a very important role in directing the growth and development of the follicle. Two oocyte-specific growth factors, namely GDF-9 and GDF-9B, have been identified in a number of species. Work in our laboratory has shown these growth factors to be very important in the early stages of follicular growth and also in dictating ovulation rate in sheep. Using in situ hybridisation, we have determined the expression patterns of these growth factors in possums and shown that they are similar to other species. One exception was the time of onset for the expression of GDF-9B. In possums, this was found to occur at an earlier stage of follicular development than has been shown in any other species to date. This earlier expression represents the possibility of targeting the entire pool of `non-growing' follicles within the ovaries, or in other words, all the eggs within the ovaries. Thus is may be possible to develop a permanent form of fertility control that that is species-specific to possums and would require only a single exposure.

In a previous study, we reported that immunisation of possums against a non-conserved region identified in each of these proteins (ie GDF-9 and GDF-9B) showed no effects on reproductive activity; suggesting these regions of the respective proteins were not important in their function. The aim of this work was to determine the effects of immunising possums against the mature region of recombinant ovine GDF-9 and GDF-9B on ovarian function.

Methods

Recombinant ovine GDF-9 and GDF-9B was produced in E. coli. The purified proteins were conjugated to keyhole limpet hemocyanin (KLH) in preparation for immunisations. Adult female possums were allocated to one of three treatment groups: GDF-9 (n=7), GDF-9B (n=7), and KLH (controls; n=6). Possums were housed in the free-range facility at Wallaceville Animal Research Centre. Male possums were present in each of the animal pens. Initial immunisations were administered using Freund's complete adjuvant (150ug protein in 0.5ml; subcutaneous). Each animal received six subsequent booster immunisations at monthly intervals (100ug protein in 1.25ml incomplete adjuvant; subcutaneous). Blood samples (2ml) were collected immediately before each immunisation. Serum was collected and stored at -20°C until assay. Three weeks after the last booster immunisation, animals were killed and their reproductive tracts examined. Blood samples were also collected at this time. Body weight and weights of the vaginal cul-de-sac and ovaries were recorded for each animal. Ovaries were fixed in Bouin's solution in preparation for histological examination.

Approach & Outcomes

Possum GDF-9B is 64% and 55% identical to the sheep nucleotide and deduced amino acid sequences, respectively. The gene structure is similar in possums to other species. The deduced amino acid sequence shows a conserved TGF_ superfamily motif following a basic proteolytic processing site. There is a potential glycosylation site in the proregion of the protein that is conserved in several species, but lacks the numerous potential glycosylation sites found in other parts of the molecule including the mature region. Moreover, as in other species, the possum sequence lacks a cysteine residue that is involved in dimer formation in other members of the superfamily.

Possum GDF-9 is 71% and 68% identical to the sheep nucleotide and deduced amino acid sequences, respectively. As in other species, an intron is located at amino acid 132 of the consensus sequence. Analysis of the deduced amino acid sequence reveals a TGF_ superfamily motif. There are three potential glycosylation sites in the sequence that are conserved across all other species reported. As was found with GDF-9B, the possum GDF-9 sequence lacks a cysteine residue that is thought to be involved in dimer formation in other members of the TGF_ superfamily.

As shown in Table 1, there was no significant effect of immunisation on body weight, cul-de-sac weight or ovarian weight. There were signs of antral follicular growth in all animals, and the number of follicles visible on the ovarian surface varied greatly.

Table 1. Mean (± sem) body weights and weights of tissues collected from possums immunised against GDF-9 or GDF-9B.

	Treatment
	Control	GDF-9	GDF-9B
Body (kg)	2.63±0.37	2.87±0.19	2.78±0.16
Cul-de sac (g)	3.77±1.81	2.33±1.34	5.47±2.32
Ovaries (g)	0.21±0.06	0.16±0.02	0.22±0.04

Discussion

GDF-9 and GDF-9B have been shown to play important roles in follicular growth and ovulation rate in several species. Gene expression studies done in our lab would suggest that these proteins have similar roles in possums. Although the patterns of gene expression for GDF-9 and GDF-9B are similar in possums to other species, immunisation against the recombinant ovine forms of these proteins failed to have a dramatic effect on the parameters observed thus far. However, it should be noted that data for two key parameters are still outstanding, namely antibody titres and ovarian histology. We have a lot of experience immunising possums against various antigens, so it is unlikely that the animals failed to generate an antibody response, but again that remains to be determined. There are two reasons for the apparent lack of effect in the current experiment: 1) GDF-9 and GDF-9B don't play important roles in follicular growth in contrast to what has been shown in other species; or 2) the structures and/or antigenic properties of GDF-9 and GDF-9B are sufficiently different between sheep and possums that antibodies directed against the ovine proteins do not affect the function of the possum proteins. If the latter is true, it may be possible to develop a permanent form of fertility control that is species-specific to possums. A subsequent study is planned to immunise possums against recombinant possum GDF-9 and GDF-9B.

5.4 PBC 255

Programme Title:	Controlled oestrus
Programme Leader:	Bernie McLeod
Institution:	AgResearch CRI

Summary

To synchronise oestrus and mating in possums to aid in the effective testing of biocontrol agents.
To characterise a possum-specific secretory channel of the intestine as a target for toxins

Objective 1: Hormonal control of follicle development.

Objective 2: Secretory mechanisms specific to the possum intestine

We have shown that in possums, secretion across the intestinal mucosa is driven by the transport of bicarbonate ions, not by chloride ions as it is in eutherian animals. The objective of this experimentation was to test whether bicarbonate ion transport, and thus secretion, across possum intestinal mucosa could be pharmacologically stimulated in vitro. To determine this we used the `channel opening' compound 1-ethyl-2-benzimidazolinine (1-EBIO). 1-EBIO has been used as a pharmacological stimulant of secretion in a number of tissues (Hamilton, Meads & Butt, 1999; Mac Vinish, Keogh & Cuthbert, 2001, Singh et al., 2001) from a wide range of eutherian mammals. Therefore, we initially assessed the effect of 1-EBIO on ion transport by the possum intestine as a test of the feasibility of this approach for a possum toxin (proof of concept).

Segments of ileum taken 10 cm from the ileo-caecal junction were cut along the mesenteric border and the underlying connective tissue and muscle layers were removed by blunt dissection. The resultant epithelial sheets were then mounted in Ussing chambers and short circuit current measured as an index of active transport.

The addition of 500 µM 1-EBIO on either the mucosal or the serosal side of the tissue, resulted in the stimulation of short-circuit current that is consistent with the stimulation of secretion. This current was insensitive to the blocking agent bumetanide, indicating that it did not involve the NaK2Cl co-transporter. It was also independent of chloride ions, but dependent on bicarbonate ions in the bathing solution. This indicates that 1EBIO was stimulating a bicarbonate ion secretory response, similar to that stimulated by the cAMP- and Ca-dependent secretagogues, forskolin and carbachol. Of some significance, pretreatment with forskolin appeared to potentiate the response to 1EBIO. The fact that forskolin, which primarily activates the apical conductance (serosal side of the tissue) of secretory tissues, potentiated the response to 1-EBIO. This suggests that the majority of the response to 1-EBIO involves stimulation of transport proteins other than the apical anion conductance mechanisms. However, this will have to be confirmed with direct measurements of the activity of this protein. In preliminary experiments, 1-EBIO failed to stimulate secretion by tissue from the colon. This is consistent with our previous demonstration that forskolin and other secretagogues do not stimulate colonic secretion in possums although they do so in eutherian animals.

References

Hamilton, K.K., Meads, L., Butt, A.G. 1999. 1-EBIO stimulates C1- secretion by activating a basolateral K+ channel in the mouse jejunum. Pflugers Arch Eur J Physiol 439:158-166

MacVinish, L.J., Keogh, J., Cuthbert, A.W. 2001. EBIO, an agent causing maintained epithelial chloride secretion by co-ordinate actions at both apical and basolateral membranes.

Pflugers Archiv-European Journal of Physiology 443:S127-S131

Singh, S., Syme, C.A., Singh, A.K., Devor, D.C., Bridges, R.J. 2001. Benzimidazolone activators of chloride secretion: Potential therapeutics for cystic fibrosis and chronic

obstructive pulmonary disease. Journal of Pharmacology & Experimental Therapeutics 296:600-611.

Background

Objective 1. The development and testing of biocontrol agents aimed at interfering with reproduction, will require the ability to synchronise oestrus and mating in possums to allow access to animals and tissues at known stage of the reproductive cycle. Standard methods used for oestrus synchronization in farm animals simply do not work in possums. In part, this is probably due to the long follicular phase in possums compared with that in farm animals, but it is becoming increasingly evident that there are fundamental differences in the hormonal control of follicle development and ovulation in possums.

As a consequence of the long follicular phase in possums, we believe that there needs to be two parts to a hormonal strategy for synchronising ovulation. The first is to synchronise the emergence of preovulatory follicles within a group of animals and the second, to synchronously induce the rupture (ovulation) of those follicles. The hormonal treatment we have been using to synchronise follicle development results in the presence of a presumptive preovulatory follicle in 75-80% of treated animals on a given day. The first objective of this programme was to assess the repeatability of this treatment to synchronise follicle development, to test the viability of these induced preovulatory follicles and to develop a protocol that will consistently induce ovulation of the induced follicles.

In previous studies, we have identified some major differences in the patterns of secretion of reproductive hormones around the time of ovulation and in the populations of receptors for these hormones in ovarian tissue. For example, there are differences in gonadotrophin concentration around the time of ovulation and there are high numbers of prolactin receptors present in possum ovaries. This suggests that there may be differences in the hormonal control of follicle development and ovulation in marsupials. We have taken two approaches to identifying such differences. Firstly, to assess mRNA expression for gonadotrophic hormone receptors in the possum ovary and secondly, to determine whether the administration of exogenous hormones at strategic times in the synchronisation protocol, will improve the incidence of induced ovulations.

Objective 2. This is based on earlier findings from our AHB-funded research into basic physiological mechanisms in the possum gastrointestinal tract. We have demonstrated that there are remarkable differences in the transport physiology (water and ion transport) of the gastrointestinal tract of the brushtail possum, compared to that of eutherian mammals. In particular, we showed that the basic mechanisms driving fluid secretion in the possum differ markedly from those found in eutherian mammals. In eutherians, fluid secretion is driven primarily by electrogenic chloride ion (Cl) transport. However, in the possum, fluid secretion is driven by electrogenic bicarbonate ion (HCO₃) transport. This suggests that a unique transport mechanism is operating in the intestinal tract of the possum (possibly in all marsupials), which could be exploited to selectively kill possums in New Zealand. Uncontrolled fluid secretion results in catastrophic water loss and rapidly leads to death. Therefore, targeting unique transport proteins to switch on secretion in the possum intestine could provide a highly-specific control strategy. This approach has significant advantages in that the pharmacological agents that activate these proteins are simple chemicals typically not subject to rapid degradation in the gut. Furthermore, they do not need to be absorbed into the bloodstream to have their effect, as the target is within the cells lining the intestinal tract itself. The basis of this objective was to characterise the secretory channels of the possum intestine with the ultimate aim of targeting these with possum-specific toxins that switch on secretion.

Objective 1: Hormonal control of follicle development and ovulation.

Introduction

In our standard synchronisation method (treating possums with oestradiol followed by an extended period of progesterone), we can synchronise follicle development with 70-80% of treated possums having have a single large (5-6mm diameter) presumptive preovulatory follicle present on Day 7 after the withdrawal of progesterone. In a series of experiments, we will assess the viability of these induced preovulatory follicles to ovulate and to produce a functionally normal corpus luteum at different times of the year. Our standard methodology is to monitor the ovarian response to the steroid hormone treatment by observing the ovaries by repeated laparoscopy following treatment, to identify the emergence of the preovulatory follicle and the time (day) of ovulation.

The primary aim of these studies was to determine what percentage of the induced preovulatory follicles would ovulate spontaneously and whether these ovulations would be normal. The second aim was to determine the expression of mRNA for pituitary hormone receptors within these induced follicles in comparison to that observed in spontaneously developing preovulatory follicles in untreated animals. The goal was to identify unusual hormone receptor expression patterns that could identify new hormonal treatments that might improve synchronisation of follicle growth and ovulation in possums. Previous investigations of antral follicle development in possums had identified some notable differences in the expression of mRNA for receptors of the pituitary hormones LH and prolactin in possums. Of particular interest, was that there was strong expression for prolactin receptors in the ovary, suggesting a possible unique role for this hormone in antral follicle development in the possum.

We have recently established a prolactin radioimmunoassay method for possums. Using this assay to analyse plasma samples collected at frequent (1h) intervals in a previous study, we found that there was transient `surge' release of prolactin that preceded the preovulatory gonadotrophin surges that occur prior to ovulation. Therefore, we carried out a series of experiments in both the breeding and non-breeding seasons to determine whether the administration of exogenous prolactin would improve the synchrony of induced ovulation in possums.

Achievement of Milestones

Determine the competency of induced follicles to ovulate spontaneously and assess luteal function of the resultant corpora lutea.

The initial series of experiments were carried out in Spring, a time when approximately 50% of possums in this region (coastal Otago) undergo oestrous cycles if not nursing pouch young. In the first study in which follicle development was monitored by repeated laparoscopy, 70% of treated animals had a single large follicle present on Day 7, but less than 10% of these ovulated spontaneously. To determine whether this was due to stress effects of the repeated anaesthesia/laparocopy, the experiment was repeated, but without any surgical intervention (laparoscopy). The endpoint monitored was the incidence of live births. No live births occurred. These experiments were then repeated in the autumn, the main breeding season in possums. Only 60% of females had a large follicle present on Day 7 after treatment and less than 20% of these follicles ovulated spontaneously.

In spontaneously ovulating mammals, the rupture (ovulation) of preovulatory follicles is triggered by a surge release of luteinising hormone (LH). In most species, a single injection of LH alone can artificially induce ovulation of the preovulatory follicle. Alternatives treatments to the use of LH, is to administer either the ovarian steroid oestradiol or the hypothalamic releasing hormone GnRH, both which will evoke an endogenous LH surge. In addition, human chorionic gonadotrophin (hCG), which has potent LH-like properties, is frequently used to induce ovulation. To our knowledge, all of these treatments will successfully induce ovulation of the preovulatory follicle in all of the species studied in which they have been used.

We have shown that, with few exceptions a single injection of LH, oestradiol, GnRH or hCG all fail to induce ovulation of large follicles in possums. This was a surprising finding as such hormonal treatments consistently induce ovulation in eutherian species. To determine whether prolactin has a direct role in ovulation, we treated animals that had a large follicle present, with ovine prolactin 3 h prior to administration of GnRH or hCG. The administration of prolactin did not increase the incidence of ovulation in either treatment group and was no more effective in the main Autumn breeding season than in Spring..

Clearly, we do not yet fully understand the hormonal requirements for preovulatory development and ovulation in possums and a better knowledge is required before we can design successful synchronization strategies.

Assess mRNA expression for LH and prolactin receptors and the steroidogenic capacity of induced follicles.

The objective of this study was to monitor mRNA expression for LH, FSH, GH and prolactin receptors in spontaneously growing follicles in possum, to try to identify the role that these hormones play in preovulatory follicle development. In an experiment aimed at determining mRNA expression associated with spontaneous follicle development, antral follicle development leading to the emergence of a preovulatory follicle was loosely `synchronized' in adult female possums by removal of their pouch young. Individual animals were euthanased at specific stages of the subsequent oestrous cycle on the basis of changes in cytology of urine samples that were collected daily, and their ovaries were recovered. The stage of the oestrous cycle was confirmed retrospectively from hormone (LH and FSH) profiles and on the appearance of the ovaries. This enabled collection of ovarian tissues at the following times;

1. Early-mid follicular phase: When animals had a number of small growing antral follicles present on their ovaries without an obvious "dominate" follicle.

2. Mid-late follicular phase: Animals had a single, large, growing presumptive preovulatory follicle present, ranging in size from 3.5 mm to > 5.0 mm diameter.

3. Early luteal phase: A single, newly-formed corpus haemorrhagicum was present, and the ovary had been recovered within 48 hours after ovulation.

4. Mid-Luteal phase: A single, mature corpus luteum was present.

The localisation of cells producing mRNA encoding for FSH, LH, GH and prolactin receptors was determined by in situ hybridisation. In addition, the expression of P450 side-chain cleavage enzyme was determined. The presence of this enzyme is used as a marker of de-novo steroidogenesis, as it is required for synthesis of any steroid. The expression of hormone receptors is summarized in Table 1.

Table 1

Structure	Receptor Expression
	LH	FSH	PRL	GH	P450scc
Small antral follicles	+++	+++	+++	++	++
Preovulatory follicles	++	-	++	++	++
Corpus haemorrhagica	++	-	-		+++
Corpora lutea	++	-	-		+++

This investigation of mRNA expression has confirmed that there are wide differences in the mechanisms that control follicle development in possums, compared to those in eutherian animals. The early expression of LH receptors in antral follicles suggests that this may have a pivotal role in the selection of the dominant follicle. The absence of FSH receptors in preovulatory follicles is surprising, again indicating a major difference in the hormonal control of antral follicle development in possums. It is clear that the interstitial tissue in possum ovaries is very active in terms of steroid hormone production, which is seldom seen in eutherian animals.

In view of the extensive differences in hormone receptor expression in possums ovarian tissues compared with that observed in eutherian species, it is not surprising that standard hormone treatments routinely used to synchronise follicle development and ovulation fail to work in the possum. Increasing our understanding of the hormonal control of follicle development in the possum is starting to unravel the mechanisms involved.

Objective 2: Secretory mechanisms specific to the possum intestine

Introduction

The bulk of the studies have focused on the use of the Ussing chamber to define the mechanism(s) of electrogenic secretion in the possum ileum. To carry out these experiments, a 10cm segment of intestine taken approximately 15cm from the ileo-caecal junction, was removed, cut open along the mesentery, and rinsed clean of luminal contents. The underlying connective tissue and muscle layers were then removed by blunt dissection and the resultant epithelial sheet mounted in an Ussing chamber. The tissues was bathed in the appropriate Ringer's on both the mucosal and the serosal sides, maintained at 37^oC and bubbled through with oxygen or with carbogen (oxygen plus carbon dioxide). Under these conditions the tissue will remain alive for a period of several hours. Electrodes attached to the mucosal tissue generate an electrical pulse in a manner that the tissue is constantly short-circuited to obtain a measure of the active electrogenic transport of ions. Transepithelial resistance (Rt) is measured at 5 minute intervals as in index of tissue permeability and viability. The system is monitored via data recorders and computer software that records all responses to treatment.

We need to determine the ion transport mechanisms involved in secretion in possums, so that we can establish that they are structurally and/or functionally different from those in eutherian mammals and birds. To some degree, this can be achieved by a process of elimination; by blocking specific ion transport mechanisms with compounds that have known effects on them and determining whether this affects the secretory process or not.

It is also possible to identify the ions involved in the function of specific ion transport mechanisms by changing the ionic composition of the Ringers solution that bathes either the mucosal or serosal side of the intestinal tissue that is mounted in the Ussing chamber. We use a range of Ringers solutions, shown in Table 1.

Table 1. Composition of various Ringers solutions used

	Ringers solutions (in mmol L^-1)
	NaHCO₃	Cl Free	HCO₃ Free	Cl/HCO₃ Free	Na Free	Na/HCO3 free

NaCl	110	0	110	0	0	0
NaGluconate	0	110	25	135	0	0
nMDGCl	0	0	0	0	110	135
KCl	5	0	5	0	5	5
Kgluconate	0	5	0	5	0	0
MgSO₄	0.5	0.5	0.5	0.5	0.5	0.5
CaCl₂	1	0	1	0	1	1
Cagluconate	0	1	0	1	0	0
NaHCO₃	25	25	0	0	25	0
Hepes/Tris	10	10	10	10	10	10
Pyruvate/glutamine	4	4	4	4	4	4
Gas	Carbogen	Carbogen	100%O₂	100%O₂	Carbogen	100%O₂
pH	7.4	7.4	7.4	7.4	7.4	7.4

Achievement of Milestones

Demonstrate that the bicarbonate dependent electrical response of the small intestine to secretagogues is due to the secretion of bicarbonate.

All experiments involved the measurement of short circuit current (Isc) as an index of the active transport of ions. Tissue was removed from distal region of the small intestine (ileum) of the animal, stripped of the underlying connective tissues and mounted as a flat sheet in an Ussing chamber. This allows selective replacement of the solution bathing either the mucosal or serosal surface of the epithelial tissue, plus the selective addition of `drugs' to either side of the tissue. The `drugs' used are all compounds that have known effects on specific ion transport mechanisms.

To confirm that the response stimulated by secretagogues was bicarbonate ion (HCO₃) secretion, we employed a combined ion substitution and pharmacological approach. Initially, tissues were bathed in solutions in which either the ions HCO₃, Cl or both HCO₃ and Cl were replaced on both sides of the tissue. The tissues were then stimulated with the appropriate secretagogues. Under these conditions, the removal of Cl resulted in a stimulation of the cAMP-dependent secretory response, whereas the removal of HCO₃ inhibited the response. The combined removal of HCO₃ and Cl did not result in any further inhibition. Similar results were obtained with the Ca²⁺-dependent secretagogue, carbachol. This clearly indicates that Cl is not involved in the secretory process in possums. Cl is the major ion driving secretion in all eutherian species studied.

We then considered the effect of asymmetric replacement of HCO₃ on the secretory responses. Tissues were either bathed in Ringer's containing HCO₃ in both the mucosal and serosal solutions, or HCO₃ was replaced with Hepes buffer in either the mucosal or serosal solution, or in both solutions. Under these conditions, the secretory response was inhibited only when the HCO₃ was removed from the serosal solution. This is consistent with the secretion of HCO₃ from the serosal to mucosal solution, as we predicted would occur.

Two potential mechanisms could be involved in the secretion of HCO₃. Given the effects of ion substitution, the most likely would involve a transporter that moves HCO₃ into the cell from the serosal solution. HCO₃ would then exit via an apical HCO₃ transporter on the luminal side of the cell. Alternatively, HCO₃ secretion could involve a serosal Na/H exchanger and an enzymic system (possibly cytoplasmic carbonic anhydrase) generating HCO₃ within the cell for secretion via an apical HCO₃ transporter, yet to be identified.

The removal of Na from the bathing solution inhibited the secretory response, confirming a role for a Na-dependent process. Specific inhibitors of the basolateral (serosal side) Na/H exchanger and of the cytoplasmic carbonic anhydrase enzyme system did not inhibit the secretion of HCO₃. This suggests that this mechanism (a Na/H exchanger and an enzyme system) was not involved in the secretion of HCO₃. Confirmation of the presence and involvement of a NaHCO₃ co-transporter in the secretion of HCO₃ in possums will require the use of other techniques. However, collectively, our studies strongly suggest that a NaHCO₃ co-transporter is involved in HCO₃ secretion in possums.

Test pharmacological compounds that may directly stimulate bicarbonate secretion.

To determine this we used the `channel opening' compound 1-ethyl-2-benzimidazolinine (1-EBIO). 1-EBIO has been used as a pharmacological stimulant of secretion in a number of tissues from a wide range of eutherian mammals. Therefore, we initially assessed the effect of 1-EBIO on ion transport by the possum intestine as a test of the feasibility of this approach for a possum toxin (proof of concept).

The addition of 1-EBIO on either the mucosal or the serosal side of the tissue, resulted in the stimulation of short-circuit current that is consistent with the stimulation of secretion. This current was insensitive to the blocking agent bumetanide, indicating that it did not involve the NaK2Cl co-transporter, one of the major ion transport mechanisms on the serosal side of secretory tissues in eutherian mammals. It was also independent of chloride ions, but dependent on bicarbonate ions in the bathing solution. This indicates that 1EBIO was stimulating a bicarbonate ion secretory response, similar to that stimulated by the cAMP- and Ca-dependent secretagogues, forskolin and carbachol. Of some significance, pretreatment with forskolin appeared to potentiate the response to 1-EBIO. This suggests that the majority of the response to 1-EBIO involves stimulation of transport proteins other than the known apical anion conductance mechanisms present in eutherian animals. However, this will have to be confirmed with direct measurements of the activity of this protein. In preliminary experiments, 1-EBIO failed to stimulate secretion by tissue from the colon. This is consistent with our previous demonstration that forskolin and other secretagogues do not stimulate colonic secretion in possums, although they do so in eutherian animals.

Approach & Outcomes

Annual Report 02/03 Summary

Objective 1

An essential requirement for the future testing of biocontrol agents aimed at interfering with reproduction will be the ability to synchronize ovulation and mating in large groups of female possums. An acceptable degree of synchronisation would be to have 80% of treated animals ovulating on a given day. To date, our synchronisation methods do not achieve this goal. The steroid hormone treatment generally results in 70-80% of possums having a single, large follicle present of a given day, but we are not yet able to induce ovulation of these follicles. We have identified major differences in hormone secretion and in the expression of hormone receptors during the period of preovulatory follicle development, in possums. Of particular note is the absence of FSH receptors in preovulatory follicles, which is essential for normal follicle development in eutherian species. The high level of expression of prolactin receptors in possum ovarian tissues and the unusual patterns of prolactin secretion immediately prior to ovulation, suggest that this hormone may play a critical role. Our preliminary attempts to mimic prolactin secretion patterns have not increased the incidence of ovulation. The inability of LH, hCG and oestradiol to induce ovulation of large follicles in possums, confirms that there are major differences in basic physiology of ovarian function in this species.

Objective 2

The research undertaken within this objective has provided further convincing evidence that secretion across intestinal epithelial tissue in possums is driven by bicarbonate ion transport, rather than by chloride ion transport. Of particular importance, is that all of our research strategies suggest that the ion transport mechanisms involved are either structurally or functionally different from those that are present in mucosal tissues of eutherian animals. A major step forward is that we have shown that it is possible to switch on secretion in possum intestinal tissue, using pharmacological agent, providing `proof of concept' of the proposed strategy for a possum-specific toxin.

5.5 PBC 256

Programme Title:	Oral bioactives
Programme Leader:	Bernie McLeod
Institution:	AgResearch CRI

Summary

To develop formulation strategies for oral delivery of biocontrol agents to possums, that maximise stability of the agent and promote its release and uptake in targeted regions of the gut.

Objective 1: Formulation for delivery to hindgut

Objective 2: Targeting specific regions of gut.

Transit studies

Data on the gastrointestinal transit of particulates and solution has been completed for 60 adult, brushtail possums (male and female). Indigestible particulates (anionic exchange resin) of different sizes were labeled with the radioiosotope technetium (^99mtc) and then orally administered to the lightly anaesthetized possum. Transit through the gastrointestinal tract was determined by gamma scintigraphy at set times after dosing. Radiolabelled fluid had the fastest transit time through the gut and there was no difference in the time taken for small (75-125 _m) compared to large (500-700 _m) particles to transit the gastrointestinal tract (figure 1). The concentration of each formulation was greatest in the caecum (the target site for delivery of biocontrol agents) 12 h after oral administration (figure 1c). Transit times of all formulations varied between individual animals.

Morphometric study

The between-animal variation in transit time raised the question of whether this could be due to differences in the morphology of the gi tract between individual possums. Therefore, the weight and length of different regions of the gastrointestinal tract (stomach, small intestine, caecum, proximal and distal colon) was measured for 26 of the animals included in the transit study. There was, however, no correlation between the lengths of different regions of the gastrointestinal tract or weights of the tissue and metabolic body weight of the animals.

Effect of feeding

Feeding and level of activity influence the transit of material through the gastrointestinal tract. To investigate this, the transit pattern over a 12 h period has been compared between possums that were dosed at night (6pm) and those that were dosed in the daytime (6am). That is, animals that were active and feeding compared with those that were inactive and not feeding. The transit of the larger particles (500-700 _m) was faster in the animals that were active and feeding compared to those that had slept for the 12h period. There was little difference between feeding and activity level on the transit of small particles or fluid.

Larger granules

The gastrointestinal transit of granules larger has also been investigated in the common brushtail possum. Granules of 1, 3 and 5 mm diameter that consisted of a core of barium sulphate and were coated with the polymers pectin and ethyl cellulose (SureReleaseTM) were orally administered to common brushtail possums. Granules 5 mm in diameter were retained in the stomach for up to 42 h and the inter-animal variability in transit time was the greatest. Particles of 3 mm diameter were located in the proximal and distal colon by 24h after oral administration, and those of 1 mm diameter were found in the caecum as well as the proximal and distal colon.

Conclusion

In order to achieve predictable and reproducible transit patterns for oral dosage forms, it is necessary to minimize the size of the particulate. For site-specific delivery to the caecum, the optimum size range of an oral dosage form is 500-700 _m and the bioactive will need to be protected during passage through the stomach and small intestine for approximately 12h before the polymer coating begins to degrade.

Background

Annual Report 02-03

We have identified the luminal and mucosal enzymes that metabolise peptides and proteins in the possum gastrointestinal tract, and have shown that the rate of degradation can be up to 1000 times lower in the hindgut (caecum and colon) than in the small intestine. Obviously therefore, the hindgut is the most appropriate region to target for oral delivery of peptide or protein biocontrol agents. The object of this programme was to develop formulation strategies to target delivery to the hindgut. This entails protecting the bioactive during its passage through the stomach and small intestine and promoting its release in the caecum and colon. To this end, we are developing tableted formulations coated with selected polymer, as well as matrix tablet that incorporate the polymers, which will protect the bioactive from degradation in the stomach and small intestine. These coatings are designed to disintegrate in the hindgut. The rates of degradation of these formulations are being assessed both in vitro and in vivo.

The successful delivery of these formulations is dependent on an understanding of gastrointestinal transit times in possums. This includes knowledge of how particle size influences the rate of passage through the intestine and the selection retention within different regions. To this end, we are carrying out an extensive programme of research to investigate transit and residence times of particulates of a variety of sizes in the possum, in order to design delivery systems that target the hindgut.

Objective 1: Formulation for delivery to hindgut

Introduction

After reviewing the literature on current polymer technology to identify products suitable for coating pelleted formulations for possums and polymers known to selectively release in the colon, pectin, chitosan and ethylcellulose were chosen as the most suitable. Tablets containing barium sulphate and a water-soluble dye were prepared, either as matrix tablets in which the polymer is incorporated throughout the tablet or as polymer-coated tablets. The range of formulations made, included some with or without inclusion of a disintegrant, which would induce rapid breakdown of the tablet once the integrity of the polymer coating was disrupted. Coating of tablets was undertaken by pan-coating to a range of coat thicknesses.

Our theory was that the pectin and ethylcellulose coating would remain intact in the stomach and small intestine, but should disintegrate in the colon or caecum. The basis for this assumption is that the microbial enzymes in the caecum and colon should degrade the pectin, thus creating a porous coat allowing the entry of water. This would lead to swelling of the granules and rupture of the polymer coat, thereby releasing the bioactive.

Coat integrity and the occurrence of breakdown of the formulations were assessed by standard dissolution testing in pH 6-7 buffer, in acid or in alkali. These were also tested by anaerobic incubation in luminal contents collected from various regions of possum intestine and by studies in the live animal.

Approach & Outcomes

Achievement of Milestones

Determine transit and disintegration of selected matrix and polymer-coated pellets in vitro and in vivo.

We initially tested matrix tablets comprising barium sulphate and fluorescein as a dye, using cellulose, chitosan and ethylcellulose as a matrix that may potentially be digested by micro-organisms of the possum hindgut. Using a variety of binding agents, these produced good matrix tablets which remained intact for at least 2 hours when incubated in buffer (pH 6.8). The initial formulations proved unstable in acid, so additional batches of the same formulation were produced and coated with Eudragit RS to give added protection from the acid environment of the stomach. These tablets were tested for rate of breakdown in luminal contents from the possum hindgut under anaerobic conditions. All of these formulations remained intact in both acid and buffer. However, their rates of disintegration in luminal contents from the possum hindgut were very slow.

We have also assessed a wide range of polymers used in tableted formulations and their characteristics were assessed both in vitro and in vivo. A high percentage of the polymer formulations tested, while suitably robust under the conditions present in the stomach and small intestine, did not show disintegration profiles under conditions of the hindgut that would be appropriate for delivery to this region of the possum gastrointestinal tract. One new formulation showed considerable promise for use in targeted delivery to the possum hindgut.

We have now extended these investigations to include a wide range of combinations and differing ratios of these polymers, to produce both coated and matrix tablets. All of these have been tested for their disintegration rates in water, in acid and alkali media and in contents recovered from the possum proximal colon and caecum - the target sites for delivery. Recently, we introduced a pre-plasticised ethylcellulose into these polymer-coating formulations. A number of the more promising polymer formulations have now been tested for degradation when exposed to luminal contents from the possum hindgut, using a viscometry to quantify degradation rates. This has identified formulations that, while being extremely resistant to degradation under the conditions encountered in the stomach and small intestine, are rapidly degraded by enzymes present in the hindgut.

In summary, these studies have determined the following points:

Viscometric experiments demonstrate that the possum hindgut bacterial enzymes degrade chitosan and the pectins.
Tablets coated with ethylcellulose alone do not disintegrate in possum hindgut contents.
Matrix tablets made with cellulose or chitosan or pectin do not disintegrate in contents of the possum hindgut:
- tablets coated with pre-plasticised ethyl cellulose alone do not disintegrate in any part of the possum gastrointestinal tract;it is difficult to coat tablets with pectin alone.

On the basis of these findings, we developed a polymer formulation that incorporates pre-plasticised ethyl cellulose and pectin that offers protection from degradation in the stomach and small intestine, but when incubated in luminal contents from the colon or caecum, the pectin is still available for enzymatic degradation. This allows for penetration of water into the tablet - leading to disintegration under conditions that would be expected in the hindgut. We have established that the ratio and source of the polymers used is critical for their suitability for colonic/caecal delivery. Tablets coated with this polymer formulation have undergone preliminary testing in vivo and proved successful. For example, although transit times through the stomach and small intestine differed by > 80% between individual possums, these formulations remained stable until they reached the hindgut where they rapidly disintegrated.

More recently, we have attempted to adopt these polymer coating formulations for smaller tablets (_ 1mm). This has proved difficult. Currently, we are experimenting with changing the content of disintegrant in the formulation. Alternative strategies would be to use matrix tablets or to develop polymer coatings where disintegration is time-dependent, rather that being based microbial breakdown.

Objective 2: Targeting specific regions of gut.

Introduction

There are two specific aims of this objective for developing formulations that will target the possum hindgut. Firstly, to assess those formulations (especially the polymer component) developed in Objective 1, for characteristics that make them appropriate for targeting the hindgut and secondly, to gain an understanding of the factors that influence transit through the possum intestine and residence times within specific regions of the intestine.

Gastrointestinal transit of solutions and of particles of various sizes through, and their selective distribution within the possum intestine, is primarily being measured by gamma-scintigraphy. Solutions or ion-exchange resins (to represent particles of different size) are radiolabeled, administered to possums and their transit through and distribution within the possum intestine is determined at various time intervals after administration. Latterly, the transit of polymer-coated formulations was determined in vivo by CT scan, or by euthanasing possums at different times after dosing and dissecting the intestine to determine the location of the tablets.

Determine stability of a peptide-lipid construct in lumen contents from possum intestine, and its uptake across intestinal mucosa.

This work is underway, but the milestone has not yet been completed. There have been problems with the analytical methods we used the lipid:GnRH construct and alternative methods have had to be developed.

The peptide-lipid construct is a novel approach used for drug delivery, where structural features of lipids are combined with those of peptides to form a chemically-conjugated molecule. These conjugates possess a high degree of membrane-like characteristics, which facilitates their passage across cell membranes. In addition, the long lipid chain of the peptide-lipid construct can offer the peptide moiety some protection from enzymatic degradation. For example, it has been shown that lipid:GnRH and lipid:TRH constructs protect the peptides (GnRH and TRH) from degradation by enzymes of the intestine in humans.

The potential for using radioimmunoassay (RIA) to measure the lipid:GnRH construct was assessed using the assay we have previously validated for possums. Using this RIA system, a comparison was made between native GnRH and the lipid:GnRH construct over a series of identical dilutions. Preliminary information indicates that the lipid interferes with antibody binding in this assay, probably due to the formation of colloidal particles. Consequently, we have abandoned the use of RIA are now investigating alternative methods of analysis, primarily by chromatographic methods (HPLC and LC-MS). We have obtained methods for extraction of the lipid:GnRH construct from samples and for the LC-MS methods which we will now use.

Establish transit and residence times of particulates in the possum gastrointestinal tract.

Studies on transit and residence times of solutions and two different sized particulates in the gastrointestinal tract, have been completed for 60 adult, brushtail possums (male and female). Indigestible particulates (anionic exchange resin) of different sizes (small, 75-125_m: large 500-700_m) were labelled with the radio-isotope technetium and then orally administered to lightly-anaesthetized possums. Transit through the gastrointestinal tract was determined by gamma scintigraphy at set times after dosing. Radiolabelled fluid had the fastest transit time through the gut, but there was no difference in the time taken for small compared to large particles to transit the gastrointestinal tract. The concentration of each formulation was greatest in the caecum (the target site for delivery of biocontrol agents) by 12 h after oral administration, but transit times for all formulations showed considerable between animal variation.

The between-animal variation in transit time raised the question of whether this could be due to differences in the morphology of the gi tract between individual possums. Therefore, the weight and length of different regions of the gastrointestinal tract (stomach, small intestine, caecum, proximal and distal colon) was measured for 26 of the animals included in the transit study. There was no correlation between the lengths of different regions of the gastrointestinal tract or weights of the tissue, metabolic body weight of the animals or transit times.

To investigate the effect of feeding and of level of activity on the transit of material through the gastrointestinal tract, possums were dosed either at night or in the daytime. The transit of the larger particles was faster in the animals that were active and feeding, compared to those that had slept over the 12h period between dosing and analysis. There was little difference between feeding and activity level on the transit of small particles or of fluid.

The gastrointestinal transit of large granules (1, 3 or 5mm) was also investigated. These granules consisted of a core of barium sulphate coated with pectin and ethylcellulose. Granules of 5mm diameter were retained in the stomach for up to 42 h and the inter-animal variability in transit time was high. Particles of 3mm diameter were located in the proximal and distal colon by 24h after oral administration.

Annual Report 02/03 Summary

Objective 1

We have developed polymer-coated tablet formulations that are robust under the acid conditions in the stomach and when exposed to the high concentrations of proteolytic enzymes in the small intestine. In addition, the inclusion of specific polymers within the coating that are degraded by microbial populations of the hindgut resulted in rapid degradation within the hindgut. However, there are a number of practical problems to be overcome when polymer-coating small particulates of 1mm diameter or less.

Objective 2

It is clear that the rate of transit of particulates through the possum gastrointestinal tract is affected by a number of factors including size of the particle, feeding and activity. Consequently, there is high variation between animals in the time taken for an orally-delivered biocontrol formulation to reach the hindgut. This emphasises the requirement for such formulations to have an extended `survival time' in the stomach and small intestine.

Publications

McLeod BJ, Thompson EG. (2002) Predation on house sparrows (Passer domesticus) and hedge sparrows (Prunella modularis) by brushtail possums (Trichosurus vulpecula) in captivity. Notornis 49, 97-101
Butt AG, Mathieson SE, McLeod BJ (2002) Electrogenic ion transport in the intestine of the common Australian brushtail possum, Trichosurus vulpecula: indications of novel transport patterns in a marsupial. Journal of Comparative Physiology B 172, 553-559
Butt AG, Mathieson SE, McLeod BJ. (2002) Aldosterone does not regulate amiloride-sensitive Na⁺ transport in the colon of the common Australian brushtail possum Trichosurus vulpecula. Journal of Comparative Physiology B 172 495-502
Wen JY, Ledger R, Davies NM, Butt AG, McLeod BJ, Tucker IG (2002) Protein and peptide degradation in the gut of the brushtail possum (Trichosurus vulpecula). Journal of Comparative Physiology B 172 495-502
Wen JY, Davies NM, Ledger R, Butt AG, McLeod BJ, Tucker IG (2002) An isocratic HPLC assay for monitoring the degradation of Luteinising Hormone Releasing Hormone by extracts from the gastrointestinal tract of possums Journal of Chromatography B: Biomedical Applications 779, 221-227
Mahoney PM, Hurst PR, McLeod BJ, McConnell MA (2002) Quantification of mast cells and microflora in the cul-de-sac of the brushtail possum (Trichosurus vulpecula). Reproduction 124, 399-408
Wen JY, Ledger R, Butt AG, McLeod BJ, Davies NM, Tucker IG (2002) Inhibition of proteolysis in luminal extracts from the intestine of the brushtail possum (Trichosurus vulpecula). Journal of Pharmacy and Pharmacology 5, 54, 1365-1372
WenJY, Ledger R, McLeod BJ, Davies NM, Butt AG, Tucker IG. (2002) Enzymatic degradation of luteinizing hormone releasing hormone (LHRH) by mucosal homogenates from the intestine of common brushtail possum (Trichosurus vulpecula) Life Sciences 71, (25) 3019-3030
Mahoney PM, Hurst PR, McLeod BJ, McConnell MA, Thompson, EG (2003) The effect of oestradiol treatment on mast cell populations and microflora in the vaginal cul-de-sac of seasonally anoestrous Brushtail possums (Trichosurus vulpecula) Reproduction 125, 733-741.
Elizabeth E Doolin, Bryce M Buddle, Bernie McLeod, Trish Mahoney, Margot A Skinner (2003) Mucosal immunity in the brushtail possum (Trichosurus vulpecula). Detection of antibody at male and female reproductive sites. Biological Procedures Online (in press)

Conference Presentations

Wen JY, Tucker IG, Davies NM, Ledger R, McLeod BJ, Butt AG (2002) The effect of absorption enhancers on the permeability of the large intestine of the Australian brushtail possum, Trichosurus vulpecula. 5^th Annual Conference on Formulation and Delivery of Bioactives. Dunedin
Fink J, McLeod BJ, Nicholson H. (2002) Identification of an oxytocin-like receptor in the prostate of the brushtail possum (Trichosurus vulpecula). Annual Conference of the New Zealand Society of Endocrinology. Christchurch.
NicholsonH, FinkJ, AssinderS, McLeodBJ, ParryL (2002). Identification and localisation of a mesotocin receptor in the prostate of the brushtail possum. Annual Conference of the Society of Reproductive Biology. Adelaide
Mahoney PM, Hurst PR, McLeod BJ, McConnell MA. (2002). Exogenous oestradiol influences mast cell populations in the vaginal cul-de-sac of the Brushtail possum Annual Conference of the Society of Reproductive Biology. Adelaide
McLeod BJ, Butt AG (2002) Possum-Specific toxins - Rule 1, know your enemy. National Possum Control Agencies National Technology Transfer Seminar Wellington.
McDowellA, McLeodBJ, TuckerIG, DaviesNM. (2002) Gastrointestinal transit of radiolabelled particles and solution in the common brushtail possum (Trichosurus vulpecula). Annual Conference of the Australian Wildlife Management Society. Sydney
Fink J, McLeod BJ, Nicholson H (2003) Seasonal changes in prostatic mesotocin and androgens in the brushtail possum (Trichosurus vulpecula) Annual Conference of the New Zealand Society of Endocrinology.
Fink J, McLeod BJ, Nicholson H (2003) Seasonal changes in prostatic mesotocin in the brushtail possum (Trichosurus vulpecula) Annual Conference of the Society of Reproductive Biology. Melbourne

5.6 PBC 257

Programme Title:	Transgenic worms
Programme Leader:	Warwick Grant
Institution:	AgResearch CRI

Summary

develop techniques that allow the production of transgenic Parastrongyloides trichosuri (a nematode parasite of possums) producing a protein(s) that interfere(s) with the growth, reproduction of longevity of its possum host.

Background

Biolistic bombardment as an alternative transformation technology.

The rationale for this objective is that although microinjection has been shown to produce transgenic P. trichosuri and that the transgenes are inherited, it is not an ideal method by which a transgenic nematode for biological control of possums could be produced. This is because, in common with the free-living nematode C. elegans, transformation of P. trichosuri by microinjection results in the formation of transgene extrachromosomal arrays composed of multiple copies of the transgene and these arrays are genetically unstable. The high transgene copy number in the arrays is a possible cause for the poor expression of the transgene that we have seen to date in P. trichosuri. Recent reports (Wilm et al. (1999), Gene 229, 31-35; and Praitis et al. (2001), Genetics 157, 1217-1226) have shown that biolistic bombardment is an alternative technique for transformation of C. elegans that results in genetically stable, low copy number transgenes that are less susceptible to gene silencing. This objective has been expanded to include electroporation as an alternative transformation technique in addition to biolistic bombardment.

a) Biolistic bombardment: We have carried out further trials of this technique with C. elegans and P. trichosuri, following the protocol described in Praitis et al. (BioRad Biolilstic PDS 1000/HE instrument, with ¼" gap distance, 9mm marocarrier to screen distance, 28" of Hg vacuum and 1350 p.s.i. rupture disk). For each bombardment, 1_l of 1_g/ml plasmid DNA was coupled to 0.6mg of 1_m diameter gold particles as described in the PDS 1000 user's manual. Approximately 10⁴ to 5 x 10⁴ worms in a monolayer on the surface of an agar plate were bombarded in each experiment.

For the initial C. elegans experiments we used the uncoordinated strain DR96 unc-76, bombarded with a plasmid containing a wild type copy of the unc-76 gene. Successful transformation would be seen as the appearance of nonUnc (wildtype) worms amongst the progeny of the bombarded worms. We carried out 8 experiments but were not successful. We therefore obtained the DP38 unc-119 strain that was used Praitis et al. The unc-119, a mutation causes a dauer defective phneotype in addition to loss of coordination. This means that in dense cultures where food is limiting (~2 generations post bombardment), unc-119 worms are unable to form dauers (and survive starvation) whereas wild type worms form dauer larvae and survive the adverse conditions. Thus transformants are selected as dauer larvae that survive starvation following bombardment. Our experience with this strain has been more satisfactory and we have experiments that await further screening to establish whether there are dauer larvae present. We also have experiments in progress to transform DP38 using microinjection (with which we have more experience) to provide a positive control for future biolistic experiments.

We have carried out 3 bombardments with P. trichosuri free-living adults. Eggs were isolated from the faeces of an infected possum by salt floatation and cultured to early adult (just prior to the onset of mating and egg production) on agar. The same bombardment conditions were used as for the C. elegans experiments except that the hsp-70 promoter-GFP-lacZ reporter construct that has shown some expression in transgenic P. trichosuri in microinjection experiments was coupled to the gold particles. Survival of free-living P. trichosuri following bombardment appeared to be similar to C. elegans (~50% mortality). The surviving worms were inoculated onto a small amount of uninfected possum faeces and cultured until the appearance of infective larvae approximately 5 days later (probably following ~2 generations of free-living growth). In the initial experiments, the larvae were collected and half were processed for _-galactosidase staining to monitor transgene expression. One worm with diffuse staining similar to that seen following microinjection was seen.

An aliquot from each of the remaining infective larval cultures was tested for the presence of the transgene by PCR of a pool of100 larvae (see figure above). One culture yielded a positive signal. When single larvae from this culture were then assayed, the frequency of the transgene was very low (<1%, data not shown) and these cultures were abandoned.

We have also continued to do some experiments (again mainly using C. elegans as a model, although some experiments with free-living P. trichosuri have also been performed) and using double stranded RNA oligonucleotides that should inhibit gene function as a test to see whether electroporation functions to at least make worms permeable to nucleic acids. We have preliminary data that this may be the case, raising the possibility that we may be able to establish a non-heritable transient expression system that will permit the rapid assessment of promoter function. We propose to test this further, since a rapid method to test putative promoters will be of great value in our search for suitable P. trichosuri promoters.

This objective remains, therefore, incomplete but we are confident that biolistic bomnardment shows considerable promise and deserves further investigation. We are also encouraged by the serendipitous finding that electroporation may provide us with a transient assay of promoter function (see objective 2).

Objective 2 DNA vectors for controlled expression

We followed two related approaches to the isolation and characterisation of new promoters to drive transgene expression, based on bioinformatic analysis of the P. trichosuri ESTs.

The two strategies were:

Design PCR primers based on the EST sequence (as close to the 5' end of the sequence as possible), PCR amplify and clone the relevant region from genomic DNA, sequence to confirm the identity of the clone and to use this clone (or the PCR product directly) to screen a P. trichosuri lambda phage genomic library. Clones from the library will be restriction mapped and likely 5' end regions isolated for further characterisation.
Probe Southern blots of genomic DNA (digested with a variety of restriction enzymes) with the PCR products from strategy 1. rather than a _ genomic library to determine if there is an enzyme that results in a fragment that contains the likely 5' end of the gene and is 3-5kb in size. The region of the gel containing this fragment will be excised, ligated into a plasmid and either re-amplified by PCR using plasmid primers or transformed into bacteria to produce a "mini-library" that can then be screened for clones containing the sequence of interest. If the PCR strategy is followed, this PCR product can then be re-cloned and thus circumvent the need to screen a "mini-library". A similar strategy was used to clone transposon tagged genes in C. elegans prior to the completion of the genome sequence.

The first of these strategies has yielded two _ clones; one for an actin gene and one for a gene we have called contig 680 and which shares some homology with aspartic protease inhibitors. The product of the contig 680 gene is likely to be an abundant secreted protein.

The two clones have been plaque purified and DNA purified for each. Digestion with NotI restriction enzyme suggests that the insert in each is ~10kb (data not shown). The correct identity of each was confirmed by verifying that it is possible to PCR amplify the probe sequence from the _ clone (see Figures above). Experiments are now in progress to determine the orientation of the gene in the clone insert and to obtain sequence in the putative promoter region of each gene.

The second somewhat less conventional strategy aimed to identify putative promoter regions by inverse PCR of genomic DNA using primers designed from EST sequences. The first step is to digest genomic DNA and determine the size of the fragments containing the probe sequences.

The status of each of the genes with which we started this process is shown in the table on the following page.

Table: current status of targeted genes

gene	observed PCR product (bp)	status	putative function & expression
act1 (actin)	500	_ clone identified	constitutive, high level, muscle
act-2 (actin)	500	PCR successful	constitutive, high level, muscle
tre-1 (trehalase)	500	Restriction fragment for inverse PCR identified	uncertain, likely to be hypodermis & constitutive
tre-3 (trehalase)	330	PCR successful	as above
laminin	380	PCR successful	hypodermis, basement membrane
bli-4 (protease)	450	Restriction fragment for inverse PCR identified	secreted, cuticle collagen processing
bli-4 (protease)	380	Restriction fragment for inverse PCR identified	as above
ben-1 (_-tubulin)	480	Restriction fragment for inverse PCR identified	constitutive, neurones
ben-1 (_-tubulin)	350	Restriction fragment for inverse PCR identified	as above
ben-1 (_-tubulin)	380	Restriction fragment for inverse PCR identified	as above
unc-14 (novel protein)	750/490		neurones
mlc-3 (myosin light chain)	no product	Restriction fragment for inverse PCR identified	muscle, high level constitutive
vti (venom trypsin inhibitor)	510	Restriction fragment for inverse PCR identified	unknown function, likely secreted protein
lev-11 (tropomyosin)	no product		muscle, constitutive
lev-11 (tropomyosin)	no product
unc-87 (calponin-like)	370	Restriction fragment for inverse PCR identified	neurones, consititutive
unc-87 (calponin-like)	420	Restriction fragment for inverse PCR identified	as above
dpy-14 (homocysteine hydrolase)	no product		collagen processing, possibly secreted from hypodermis
dpy-19 (novel protein)	1500	PCR successful	hypodermis & some neurones
contig 9	no product
contig 16 (aspartic protease inhibitor)	cloned in previous work	_ clone identified	unknown, but probably secreted

Thus the promoter objective has reached a point where we have two _ clones and several candidates for inverse PCR. This is expected to yield at least one muscle and one secreted promoter (from the _ clones) and perhaps additional muscle, secreted and neuronal promoters in the next few months.

The possibility that electroporation may provide a route to transient expression testing of these, and the construction (in other work) of a set of Gateway promoter/reporter vectors means we will be able to rapidly test the promoters as they become available.

Objective 3 Transgenic worms in possums in containment

This objective has been delayed by two factors. First, it has taken some time to obtain first ERMA approval to infect possums with transgenic worms and then MAF approval of a containment facility. Second, our intention was to use either stable biolistic transformants or, at the very least, microinjection transformants showing improved levels of _-galactosidase expression.

We have ERMA and in principle MAF approval (contingent on minor modifications of the facility). More importantly, we have a stock of infective larvae from a microinjection experiment where the frequency of worms expressing the _-gal reporter and the levels of expression were much higher than in any previous experiment. These larvae will soon be put into possums and the production of transgenic progeny assayed in the faeces of the infected possums.

Approach & Outcomes

This has been a challenging year in which a combination of science and regulatory difficulties has prevented us from meeting all of our milestones. We have, however, made progress towards each. In particular, we have consolidated our ability to make transgenic P. trichosuri by microinjection to the point where we have obtained significantly improved expression of the hsp-70 _-galactosidase reporter and it is sensible for us to proceed with infecting a possum with these worms. We have also made considerable progress towards identification and testing of new promoters derived from a range of genes with a range of likely expression levels and tissue specificities. The observation that electroporation may serve as a route to a rapid, transient expression test of promoters (and the concurrent development of Gateway promoter vectors) has set the stage for rapid testing of these and other promoters as they become available.

Confirmed biolistic transformation of either C. elegans or P. trichosuri has eluded us so far, but we were unable to start on these experiments due to delays with ERMA approval. We have obtained preliminary evidence (one stained larva) of transformation in P. trichosuri with this method and have obtained and are now testing a more suitable strain of C. elegans. We have also made arrangements to visit an Australian laboratory which has recently established biolistic transformation of C. elegans (at the ANU in Canberra) to obtain some firsthand experience of the technique. We remain optimistic, therefore, that we will be able to develop biolistic transformation for both species.

5.7 PBC 258

Programme Title:	Bovine Tb
Programme Leader:	Bryce Buddle
Institution:	AgResearch CRI

Summary

and evaluate diagnostic tests and vaccines for control of bovine tuberculosis in cattle and possums. Develop and delivery for measuring mucosal immune response in possums and delivery of vaccines for biocontrol of possums

Background

Objective 1 Immunology of bovine tuberculosis

a) Improved diagnostic tests and vaccines for control of bovine tuberculosis

Description

Develop and evaluate diagnostic tests and vaccines for control of bovine tuberculosis in cattle and possums.

Establish whether the addition of dead BCG to an encapsulated oral bait vaccine lowers the effectiveness of the live BCG vaccine for possums and determine the optimal amount of live BCG in an oral bait for protection against tuberculosis.
Compare the efficacy of a newly derived attenuated Mycobacterium bovis strain to BCG for protection of possums against bovine tuberculosis when the vaccines are administered as an oral bait.
Determine which possum gut associated lymphoid tissues are sensitised to M. bovis antigens following administration of oral bait BCG vaccine, with the aim of optimising the uptake of BCG in the gut where the delivery of BCG can be altered by varying the encapsulation process.
Plan a field trial to determine the efficacy of an oral bait BCG for wild possums.
Determine whether the addition of zinc ions to tuberculin increases the sensitivity of the tuberculin skin test response in M. bovis-infected animals.
Compare the effectiveness of an ESAT-6/CFP10 interferon-_ test with the standard interferon-_ test for the differentiation of cattle infected with M. bovis and those exposed to environmental mycobacteria (part funded by AHB).
Identify specific host genes from peripheral blood mononuclear cells (PBMC) of M. bovis-infected cattle, which are up or down-regulated when re-exposed to M. bovis antigens. Analyse gene expression using DNA micro-arrays on total PBMCs.
Complete the study to identify adjuvants and immunostimulants which induce strong cellular immune responses to M. bovis antigens in cattle and evaluate the best combinations of these in vaccines for protection against bovine tuberculosis (funding predominantly from DEFRA, UK).
Submit three papers to refereed journals.

b) Possum Immunology

Description

Develop methods for measuring mucosal immune response in possums and identify vaccine delivery systems for biocontrol vaccines.

Develop a serological test to measure immune responses of possums to a possum specific nematode which may be used as a vector for a biocontrol vaccine (collaboration with Mark Ralston).
Determine the immune responses and hormone levels in possums following vaccination with a conjugated GNRH vaccine (collaboration with Ken McNatty and Doug Eckery).
Test the effectiveness of including adjuvants and immunostimulants into a zona pellucida vaccine to enhance immune responses following oral delivery topossums (collaboration with Janine Duckworth and Phil Cowan).
Submit one paper to a refereed journal.

Summary

High (10⁹) and medium (10⁸) doses of oral BCG were more immunogenic for possums than a low (10⁷) dose of oral BCG. A further trial comparing the protective efficacy of a high and medium dose of oral BCG is in progress as is a trial comparing live and dead BCG with live BCG.
A comparison of two newly derived attenuated strains of M. bovis showed that WAg530 was more immunogenic when delivered by the oral route than WAg533. A trial is in progress comparing the protective efficacy of a newly attenuated M. bovis strain with BCG when the vaccines are delivered as oral baits.
Possums which consumed a single oral bait containing 10⁸ CFU of BCG shed BCG in their faeces for a brief period (between 1 to 7 days) after vaccination and the number of BCG shed in the faeces was relatively low. Mucosal immune responses to M. bovis antigens were detected in a small proportion of possums up to 38 days after consumption of an oral bait BCG vaccine. Further possums have been vaccinated to study immune responses at 56 days after vaccination.
A plan for a field trial of oral bait BCG vaccination of possums has been established and is under review by the Animal Health Board.
Application of zinc in the form of a crème to the skin test site does not increase sensitivity of the tuberculin skin test in M. bovis infected cattle.
A field diagnostic trial indicated that the ESAT-6/CFP10 interferon-_ test was more specific for the diagnosis of bovine tuberculosis than the standard interferon-_ test, but some tuberculosis cattle were not detected using the ESAT-6/CFP10 test. The best application for this new test is for confirmation of tuberculosis in areas where false positive reactions are common, but these areas are generally free of tuberculosis (i.e. Northland, Waitako and Taranaki).
Methods have been developed for measuring host gene expression from peripheral blood lymphocytes of M. bovis-infected cattle using DNA micro-array technology.
Inclusion of CpG oligdeoxynucleotides into a protein subunit vaccine induced enhanced protective Th1 immune responses and significant protection of cattle against an experimental M. bovis infection after protein subunit vaccination.
Antigen preparations suitable for using in a serological test for immune responses against a possum specific nematode, Parastrongyloides trichosuri (PT) have been identified. An ELISA using these antigens gave positive results with serum of possums from areas where PT is present and gave negative results with serum of possums from a PT free area.
Antibody responses to GnRH after vaccination of possums with a conjugated GnRH vaccine correlated with the effect that vaccination had on reproductive hormone levels.
Mucosal delivery of a zona pellucida vaccine (ZP3) to possums, in combination with BCG, stimulated specific antibody at reproductive sites but not serum antibody nor cell-mediated responses to ZP3.

Approach & Outcomes

Establish whether the addition of dead BCG to an encapsulated oral bait vaccine lowers the effectiveness of the live BCG vaccine for possums and determine the optimal amount of live BCG in an oral bait for protection against tuberculosis.

Compare the efficacy of a newly derived attenuated M. bovis strain to BCG for protection of possums against bovine tuberculosis when the vaccines are administered as oral bait.

Delivery of tuberculosis vaccines as oral baits is a practical means of vaccinating wild possums against tuberculosis. A recent break-through has resulted from work by Frank Aldwell, Otago University where live BCG has been encapsulated in a lipid matrix. This encapsulation has prolonged the shelf-life of the vaccine to 12 weeks at 4_C and protects the BCG from degradation in the stomach of animals. Studies on the shelf-life and efficacy studies in mice were recently published and were carried out in-conjunction with our group at Wallaceville (Aldwell et al., 2003). Studies with oral bait BCG vaccine in possums have shown that the level of protection achieved with this vaccine was similar to that achieved with BCG administered by the subcutaneous route (Aldwell et al., submitted). In the field, vaccine baits may not be treated optimally and some of the BCG may die before possums eat the bait. It is important to establish whether the inclusion of dead BCG in a vaccine bait will induce an adverse effect. Secondly, the optimal amount of live BCG in a bait needs to be determined. Thirdly, there is a need to determine whether other attenuated M. bovis strains protect possums better than BCG when administered in an oral bait.

Two trials were undertaken to address these questions. In the first trial, 36 possums, which had been captured from the Upper Hutt area, were divided into six groups (6 animals/group) with each group having similar numbers of males and females and similar mean body weights. All possums were acclimatised to the cages for at least 3 weeks before vaccination and no possums had peripheral blood lymphocyte proliferation assay (LPA) responses to bovine PPD of > 3 (stimulation index, SI). Possums from two groups were given an oral tablet containing either 10⁸ colony forming units (CFU) of WAg530 or WAg533 and the treatments were repeated the next day. WAg530 and WAg533 are newly derived attenuated M. bovis strains developed by Des Collin's group at Wallaceville. Possums from three other groups were given a tablet containing 10⁷, 10⁸ or 10⁹ CFU of BCG and possums from a sixth group were given a placebo tablet. The treatments were repeated the next day. The possums readily ate the tablets. All of the possums were challenged with an aerosol of M. bovis using the aerosol generating chamber 7 weeks after vaccination and were killed and necropsied 7 weeks after challenge. Blood samples were collected for the LPA immediately prior to vaccination, at 4 and 7 weeks after vaccination and at 4 weeks after challenge. Stimulation indices were calculated for responses to avian and bovine PPD (purified protein derivative prepared from Mycobacterium avium and M. bovis respectively) and to a mitogen Concanavalin A (ConA).

The second trial was set up in a similar manner. Forty male possums captured in the Upper Hutt region were screened in the LPA assay to ensure they were all negative for responses to bovine PPD (all SIs < 3.0). These animals were divided into six groups (6-7/group). Possums from the first group (n=7) were each fed an oral bait containing 2 x 10⁸ CFU of live BCG (medium dose group) on two consecutive days. Possums from the second (n=7) and third (n=6) groups were fed oral baits in a similar manner with the baits containing 1 x 10⁹ CFU of live BCG (high dose BCG) or 2 x 10⁸ CFU of live BCG/ 8 x 10⁸ CFU of dead BCG (live/dead BCG), respectively. Possums from a fourth group (n=7) received oral baits containing 1 x 10⁹ CFU of live newly attenuated M. bovis strain. Possums from the fifth group (n=6) were vaccinated subcutaneously with 10⁶ CFU of live BCG to act as a positive control and the sixth group (n=7) were not vaccinated to act as a negative control. Blood samples were collected from the animals for the LPA at 5 weeks after vaccination and the possums will be challenged with virulent M. bovis on 26 June 2003.

In the first trial, there was an indication that the animals had been presensitised to mycobacterial antigens as the animals in three of the vaccinated groups (medium dose BCG, high dose BCG and WAg530 groups) produced strong LPA responses to bovine PPD (stimulation index, SI > 5) at 4 weeks after vaccination. This is several weeks earlier than we had previously observed strong LPA responses in possums that have consumed oral BCG vaccine baits. Secondly, the remaining groups produced strong responses at 7 weeks after vaccination (immediately prior to challenge), with 4 of 6 of the placebo-vaccinated possums producing SIs to bovine PPD ranging from 10-58. The parameters used to assess vaccine efficacy included body weight loss between challenge and necropsy, lung weight as an indication of extent of lung lesions and bacterial counts from the lungs and spleen. Although, there were no significant differences between the groups, the control group had the highest mean loss in body weight and spleen bacterial count as well as the second highest lung weight and lung bacterial count. The lack of significant differences between groups may have been in part due to the high variability within groups, which in turn may have been due to sensitisation of the possums, prior to challenge, to mycobacteria or cross-reactive antigens. This is the first time we have seen strong LPA responses to bovine PPD in negative control possums prior to challenge in the 12 years of conducting immunological studies in possums. Even though the apparent sensitisation may have interfered with protective outcome, the results of the immunological studies at 4 weeks after vaccination indicated that the high and medium doses of BCG were more immunogenic than the low dose BCG group and WAg530 was more immunogenic than WAg533.

In the second trial, the LPA responses to bovine PPD followed the pattern we have seen in the majority of studies. Four of the six subcutaneously BCG-vaccinated possums produced LPA responses to bovine PPD (SI > 4.0) at 5 weeks after vaccination, while none of the oral bait-vaccinated or non-vaccinated possums produced responses SI > 3.4. It is expected that the majority of the oral bait-vaccinated possums will produce LPA responses to bovine PPD at 8 weeks after vaccination (26 June) when the possums will be challenged with virulent M. bovis. The possums are scheduled to be killed and necropsied at 7-8 weeks after challenge.

Faecal shedding of BCG and mucosal immune responses of possums following consumption of oral bait BCG vaccine (part funded by AHB).

To obtain regulatory approval for the use of oral bait BCG vaccine for possums, information is required on shedding of BCG in the faeces and the persistence of BCG in the tissues of the possums that have consumed oral bait BCG vaccine. The current study was designed to answer questions relating to shedding and persistence of BCG in possums and to investigate the induction of mucosal immune resonses to orally administered BCG. Twelve male possums were captured from the Upper Hutt region and housed individually in cages at Wallaceville. Blood samples collected from these animals prior to vaccination produced negative lymphocyte proliferation responses to bovine PPD (all SIs < 3.0). A single oral bait vaccine (1 g) containing pelleted BCG Pasteur 1173P2 resuspended at a concentration of 1 x 10⁸ CFU in lipid C, plus bait attractants (glucose and anise oil) was fed to each of eight possums (BCG-vaccinated group). An oral bait prepared in a similar manner, but not containing BCG was fed to each of the remaining four possums (control group). The possums were checked to ensure all the animals had eaten the baits. Faecal samples were collected from the cages of the vaccinated possums prior to and at 1, 2, 3, 7, 14 and 21 days after vaccination and from the control possums prior to and at 1 and 3 weeks after consumption of the placebo bait. At 21 days after feeding the oral baits, 4 vaccinated and 2 control possums were killed. At 38 days after feeding baits, faecal samples were collected from the remaining possums and these animals were then killed. After the animals were killed, tissues including Peyer's patches (lymphoid tissue from the small intestinal tract), mesenteric lymph nodes, spleen, liver and lungs were collected for bacteriology. Heparinised blood samples were collected from all of the possums to measure lymphocyte proliferation responses to bovine PPD prior to and at 21 days after feeding the oral baits and at 38 days after vaccination for the remaining 4 vaccinated and 2 control possums.

The mean number of BCG recovered from the faeces of the vaccinated possums over the first 14 days and the proportion of animals from which BCG was isolated is shown in Figure 1. When no BCG was isolated from the faeces, a count representing half the minimal count 5 CFU/g of faeces (0.699 log₁₀ CFU/g) was recorded. In the vaccinated animals, no BCG was isolated prior to vaccination and at 14, 21 and 38 days after vaccination. The highest mean number of BCG shed was at 2 days after vaccination and highest number of BCG shed by an individual possum was 10⁴ CFU/g of faeces. No BCG was isolated from the faeces from the non-vaccinated animals. Tissue samples from possums killed on days 21 and 38 days after vaccination have been cultured, but results are currently not available. In summary, possums which consumed a single oral bait containing 10⁸ CFU of BCG shed BCG in their faeces for a brief period (between 1 to 7 days) after vaccination and the number of BCG shed in the faeces was relatively low.

We have previously developed methods for determining sensitisation to mycobacterial antigens in possums by measuring responses of peripheral blood lymphocytes using lymphocyte proliferation assay (LPA). This has been used to determine the level of sensitisation to purified protein from M. bovis (bovine PPD) after possums have eaten oral bait formulated BCG (see MAF report June 2002). In order to understand more fully how oral bait formulated BCG stimulates protective immune responses in the possum, protocols were developed for using LPAs to measure cell-mediated immune responses in gut associated lymphoid tissue. Responses from draining lymph nodes and Peyer's patches were optimised with respect to preparation of lymphoid cells from these tissues and in vitro culture conditions. Lymphocytes from Peyer's patches proximal to the stomach gave stimulation indices (SI) of >10 in 9/10 possums in response to the T cell mitogen concanalin A and in 6/10 possums in response to the B cell mitogen of pokeweed. When lymphocytes were derived from distal Peyer's patches 6/10 and 4/10 possums responded in this way to the T cell and B cell mitogen respectively. A series of experiments were then carried out to measure cell-mediated responses in gut associated lymphoid tissue at various times after possums were given oral bait BCG. Groups of possums (n= 6-7) were given lipid formulated oral BCG bait containing 10⁸ CFU BCG or lipid formulation alone. All possums ate the bait. They were sacrificed 3, 7, 21 or 38 days later and immune responses to bovine PPD of blood and splenocytes were compared with those of mesenteric lymph node cells and lymphocytes from a mixture of proximal and distal Peyer's patches. None of the possums given lipid formulation alone gave a response of SI>3 at any time point and neither did possums given BCG and sacrificed after 3 or 7 days. A positive cut-off for LPA has been established as SI _ 3.4 from taking the mean response of animals prior to vaccination and adding 3 standard deviations of the mean. Possums that gave a SI >3.4 at 21 and 38 days are shown in Table 1. At 21 days 2/7 vaccinated possums gave spleen and mesenteric lymph node responses but responses were still only detected in a proportion of the possums at 38 days. A response from Peyer's patches was detected in one vaccinated possum at this time. The results show that it takes some weeks to generate cell-mediated immune responses to BCG given in bait formulation and responses in mesenteric lymph nodes and Peyer's patches are able to be measured but do not necessarily correlate with responses from blood. An experiment is in progress to monitor immune responses at 56 days after vaccination as results from other work suggest optimal responses after consumption of oral bait BCG may take some time to develop.

Table 1. Number of possums responding to bovine PPD after eating BCG oral bait.

	Killed at 21 days				Killed at 38 days
	Blood	Spleen	MLN	PP	Blood	Spleen	MLN	PP
BCG in lipid	0/7	2/7	2/7	0/7	4/7	4/7	2/7	1/7
Lipid alone	0/4	0/4	0/4	0/4	0/4	0/4	0/4	0/4

Positive response SI >3.4

MLN, mesenteric lymph; PP Peyer's patch

Field trial to determine the efficacy of an oral bait BCG for wild possums.

A field trial carried out by Leigh Corner from Massey University has shown that possums vaccinated both by an intranasal aerosol using a hand-held applicator and by intraconjunctival instillation had a lower incidence of natural M. bovis infection than non-vaccinated possums. Over the two years of the study 300 possums were recruited to the study with 149 being allocated to the vaccination group. There were significantly fewer cases of tuberculosis in the vaccinated (4 cases) than in the non-vaccinated group (13 cases; P=0.023). However, there were some problems with the study in that for a vaccine to work in the field it must be self administered to possums and to-date this has not been achieved with an aerosol vaccinating device. Secondly, there was a marked decline in possum numbers on the study site over the 2 year period, which may have led to a decline in the number of tuberculosis cases. Oral bait BCG can be self administered to possums and it is now at a stage where it can be tested in a field trial. The Animal Health Board has asked Dave Ramsey from LandCare and investigators at AgResearch Wallaceville to design a field trial to test the efficacy of oral bait BCG in the field.

The study will be conducted at two sites with a known history of tuberculosis infection. Depending on the prevalence and/or incidence of disease, these sites are likely to be in the Wainuiomata area, which is currently not subject to vector control operations. Sites will consist of a grid of approximately 200 cage traps set at 30 m spacing. Initial trapping will be undertaken at each site to determine disease prevalence and distribution. All traps will be baited with apple lured with flour and aniseed with trapping sessions occurring over 4 consecutive nights. During each trapping session, all captured possums will be anaesthetised with an injection of ketamine hydrochloride at an average dose of 35mg/kg. Once sedated, possums will be individually marked with a metal ear tag and tattoo and standard morphological measurements taken such as weight (g), head/body and total length (mm). The tuberculosis infection status of all captured individuals will be ascertained both by palpation of the major superficial lymph nodes as well as the use of the lymphocyte proliferation assay (LPA). LPA tests will be carried out by staff at AgResearch, Wallaceville. From possums with suspect lesions, swabs, in the case of open lesions, and aspirated fluid using a sterile needle for closed lesions will be used to collect samples for bacteriological confirmation of infection.

Prior to the application of any vaccination treatments, the likely force of infection in each population will be estimated using data on age-specific prevalence of tuberculosis (Caley and Hone, 2002). Possums will be assigned to an age class based on tooth wear characteristics. The estimated force of infection will be used in a stochastic stage-structured Leslie matrix model with stages representing susceptible and infected age classes of possums to predict the likely incidence of disease in non-vaccinated possums over a 2 year time period. This information will then be used in a power analysis (Skalski and Robson 1992) to determine the minimum size of any treatment effect (difference in the force of infection) between vaccinated and non-vaccinated individuals that has an adequate probability of being detected. This will invariably be dependent on both the existing force of infection and population size.

If adequate power exists, then the requisite number (estimated from the power analysis) of randomly selected uninfected individuals of both sexes will be subject to vaccine treatments. Treatment individuals will be considered for selection if they are considered to be `at-risk' of encountering diseased individuals based on trap-revealed home range overlap. Each treatment individual will be fed one oral BCG vaccine bait, equivalent to one effective dose by offering the bait to the trapped possum. Where possible, all treatment individuals will be matched with one or more `control' uninfected individuals. Individuals will be matched on the basis of sex, sexual maturity, condition and home range use.

Following the application of vaccination treatments, trapping will continue at each site at bimonthly intervals. To determine the presence of tuberculosis infection at each trapping occasion, all captured treatment and control individuals will be subject to palpation and blood samples would be collected for LPA from up to 60 possums/ bimonthly interval. Possums with a positive LPA sample will be fitted with a mortality sensing radio collar at the next bimonthly period to ensure they can be monitored more closely for signs of clinical disease. Trapping will continue for a minimum of 2 years. However, results for the trial will be reviewed after 6 months to determine whether the tuberculosis incidence rate in uninfected individuals is in line with predictions.

Assuming that the 6 monthly results are satisfactory, routine trapping will continue and tuberculosis testing in both vaccinated and control animals for a further 18 months. Mortality rates of vaccinated and control animals will be estimated during this period and extra animals will be added into either group to maintain sample size. This `staggered entry' of subjects into the experiment can be catered for in the analysis. During the final trapping occasion, all captured animals will be humanely killed and necropsied for signs of clinical tuberculosis. Pooled lymph nodes from each necropsied animal will be submitted for bacteriological confirmation of their tuberculosis infection status.

The incident cases of tuberculosis infection (based on LPA sampling) in both treatment and control groups of individuals will be analysed to estimate the instantaneous incidence of disease (force of infection or hazard rate). The incident cases of clinical disease (based on palpation and necropsy) will also be analysed to estimate the hazard rate for clinical disease. For both infection and clinical disease, the difference in the magnitude of the hazard rate will be used to estimate vaccine efficacy. The vaccination could start in December 2003 and final trapping is estimated for December 2005. It is expected that bacteriology results will not be available before the end of March 2006 and final report will be completed by June 2006.

AgResearch Wallaceville will contribute to this study in the planning of the trial, administration of the vaccine baits to the possums, collection of blood samples from possums, carrying out LTA assays, necropsy of animals and analysis of results. Work undertaken by AgResearch is proposed to be funded from our current MAF Policy grant and if the trial proceeds, LandCare will be funded by AHB. The AHB are in the process of sending this proposal to referees for review.

Determine whether the addition of zinc ions to tuberculin increases the sensitivity of the tuberculin skin test response in M. bovis-infected animals.

In some areas of New Zealand the tuberculin skin test has a poor sensitivity in detecting Mycobacterium bovis-infected cattle. Some reports dealing with human tuberculosis have indicated that zinc deficiency may be associated with weak tuberculin skin test responses. The application of zinc crème to the skin test inoculation site immediately after the inoculation of tuberculin in humans and guinea pigs has resulted in enhanced skin test responses at 48-72 hours compared to responses at skin test sites where the zinc crème was not applied. These results have raised a query as to whether skin test responses in cattle could be enhanced when zinc crème was applied to skin after inoculating tuberculin. Originally, we had considered adding zinc salts to tuberculin, however inoculation of zinc salts in vivo can cause an inflammatory response and this approach was not pursued.

Three experiments were undertaken to determine the effect of zinc crème on tuberculin skin test responses. The experimental design was the same in the three studies. An area on both the left and right sides of the neck was shaved and in each shaved area 0.1 mls of avian and bovine tuberculin was inoculated at sites approximately 8 cm apart. Zinc crème (7.5% ZnO in 50% castor oil) was applied to inoculation sites on the right side of the neck. Skin fold thickness at the inoculation sites was measured prior to inoculation and 72 hours later. Skin testing was carried out on:

5 non-infected calves - experiment 1.
20 non-infected calves, 10 of which had been vaccinated with BCG 10 weeks previously - experiment 2.
11 M. bovis-challenged calves of which 5 had been vaccinated with BCG - experiment 3.

The results of the skin testing are shown in Table 2.

Table 2. Mean increases in skin-fold thickness at 72 hours (mm)

	Avian tuberculin	Bovine tuberculin	Avian tuberculin + Zn	Bovine tuberculin + Zn
Non-infected (5)	0	0	0	0

Non-infected (10)	1.0	0.3	1.0	0.7
Non-infected, BCG-vaccinated (10)	4.0	5.6	3.4	5.6

M. bovis-challenged (6)	1.5	12.7	1.8	11.4
M. bovis-challenged, BCG-vaccinated (5)	2.8	6.4	3.1	8.3

There were no significant differences in the mean skin-fold thicknesses between the sites where zinc had been applied and the control sites. In all three experiments, there was a heavy fall of rain within the first 24 hours after application of the zinc crème and this may have washed off the crème. An alternative view was that the calves were not zinc deficient and application of the zinc had no effect, or application of zinc crème has no effect on tuberculin skin test responses in cattle. It was of interest to note that the tuberculin skin test responses of the M. bovis-infected group that had not been vaccinated were significantly greater than the equivalent group that had been vaccinated with BCG (P<0.05), suggesting that BCG-vaccination had reduced the extent of the M. bovis infection. No further studies are currently being planned for application of zinc crème to tuberculin skin test sites. If a cattle herd is found where the tuberculin skin test is not identifying M. bovis-infected animals, blood samples will be taken from the animals to determine if they are zinc deficient.

Use an ESAT-6/CFP10 IFN-_ test to differentiate cattle infected with M. bovis from those exposed to environmental mycobacteria (part funded by AHB).

Preliminary results from a MAFPolicy/AHB funded trial in 2000/01 showed that a combination of two M. bovis antigens (ESAT-6 and CFP10) used in the IFN-_ test markedly enhanced specificity compared to the standard IFN-_ test which uses avian and bovine PPD. An application for this new test would be in a tuberculosis endemic area, which has a history of non-specificity skin test reactivity (West Coast of the South Island). A project was started in 2002 to determine if an improved IFN-_ test could be developed with enhanced specificity for detection of M. bovis infection. Blood samples were collected from cattle, which had reacted positively in the skin test in the last 10-30 days. The bloods were assayed using the standard IFN-_ test as well as with the ESAT-6/CFP10 antigen combination.

Blood samples were submitted from 184 cattle from 40 herds on the West Coast, 5 animals from one herd in Southland and 20 animals from three herds in the Waikato, with all cattle reacting positively in the caudal fold test in the previous 10-30 days. Animals that reacted positively either in the standard IFN-_ test (cut-off: Bovine PPD OD - Avian PPD OD _ 0.100) or the ESAT-6/CFP10 assay (cut-off: ESAT-6/CFP10 OD - Nil OD _ 0.035) were killed and examined by meat inspectors for tuberculous lesions. It was requested that samples from tuberculous lesions be collected for bacterial culture and from non-lesioned animals, a pool of retropharyngeal, bronchial and mediastinal lymph nodes be collected for culture of M. bovis. A total of 33 animals were killed, but unfortunately samples for bacterial culture were only collected from 10 of these animals. This has made the interpretation of the results very difficult as without culture we cannot confirm whether the animals were M. bovis-infected. Alternatively, tuberculous lesions can be very difficult to detect macroscopically in cattle and it is not uncommon to culture M. bovis from a pool of lymph nodes from non-lesioned "suspect" animals.

A total of 24 animals from the West Coast reacted positively in the standard IFN-_ test and 9 of these had suspect tuberculous lesions. The initial cut-off for the ESAT-6/CFP10 appeared to be too low and 5 animals with a differential OD value between 0.035 - 0.038 had no lesions. The cut-off was amended to _ 0.040 and a total of 12 animals reacted positively to ESAT-6/CFP10 using this criterion. Six of these had suspect lesions (5) or M. bovis was cultured from a pool of lymph nodes (1). The differential ESAT-6/CFP10 OD value for these animals ranged from 0.72-0.217, while for the 6 apparent false positives, the OD value ranged from 0.043-0.553. The standard IFN-_ test missed one animal, which had no lesions but was culture positive for M. bovis, while the ESAT-6/CFP10 test missed four suspect tuberculous lesioned animals. There were 13 apparent false positive reactions in the standard IFN-_ test and 6 apparent false positive reactions in the ESAT-6/CFP10 test (cut-off _ 0.040).

The 5 animals from Southland were negative in both IFN-_ tests. In one Waikato herd 5 of 9 animals reacted positively in the standard IFN-_ test, but were all negative for the ESAT-6/CFP10 test. Slaughter of one animal and subsequent skin testing of this herd revealed that this herd did not have bovine tuberculosis. In the second herd all 8 of the caudal fold test positive animals reacted positively in the standard IFN-_ test and 5 of 8 reacted in the ESAT-6/CFP10 test. Four of these animals had macroscopic tuberculous lesions when examined after slaughter, but no samples were collected from these animals to confirm which animals were culture-positive for M. bovis. A further 3 animals from the third Waikato herd were negative in both IFN-_ tests.

In summary, results from this study must be interpreted with caution as the infected status of individual animals was not confirmed in the majority of the test positive animals. Even though clear instructions were provided to field personnel, the appropriate samples were not always collected. The study has shown that the ESAT-6/CFP10 test can reduce false positives, but does miss some tuberculous animals. This test has particular application in areas of New Zealand, which are considered free of tuberculosis, and there are numerous animals producing false positive reactions in the standard IFN-_ test. The cut-off for the ESAT-6/CFP10 test should be amended to ESAT-6/CFP10 OD - Nil OD _ 0.040 rather than _ 0.035.

Identify specific host genes from peripheral blood mononuclear cells (PBMCs) of M. bovis-infected cattle, which are up or down-regulated when re-exposed to M. bovis antigens. Analyse gene expression using DNA micro-arrays on total PBMCs.

The use of unique DNA microarray technology has the potential to identify host genes and proteins, which play an important role in either protective immunity to tuberculosisor disease. We have used DNA microarrays, developed as part of an AgResearch gene discovery initiative, to profile bovine PPD-specific gene expression in PBMCs from M. bovis-infected cattle.

The first experiment was aimed to determine whether the DNA microarrays could successfully be used to identify genes whose expression is known to be up regulated during infection with tuberculosis. PBMCs were isolated from two tuberculosis infected cattle 12 weeks after challenge with M. bovis and cultured for 20 hr with either PBS or bovine PPD. Both animals gave strong IFN-_ responses to bovine PPD in the whole blood IFN-_ assay conducted at 10 weeks after challenge. Purified RNA was prepared from both stimulated and non-stimulated PBMCs, labelled with either Cy3 or Cy5 fluorescent dyes and hybridised to DNA microarrays in triplicate. These microarrays were printed with 12,000 ESTs and contained, in addition a number of PCR products encoding relevant immunological genes such as cytokines and chemokines. The slides were scanned at Otago University and images converted to data using GenePiz software analysis program. They were analysed by bioinformatics to identify genes up-regulated or down-regulated in expression in response to antigenic re-stimulation. This approach identified a number of differentially regulated genes and in particular, showed that expression of both IFN-__and IL-2 in PBMCs from the tuberculosis infected cattle was increased following antigenic re-stimulation. These results indicate these DNA microarrays can distinguish key immune genes known to be involved in tuberculosis, and potentially the microarray procedure may identify novel genes important in immunity to tuberculosis.

A second experiment was conducted to profile the pattern of gene expression in PBMCs from cattle during the time course of tuberculosis infection following challenge with M. bovis. Three non-vaccinated animals, from the vaccine/adjuvant cattle trial which had low IFN-_ responses to bovine PPD prior to challenge were used. PBMCs were isolated from these animals pre-challenge and at 2, 5, 9 and 12 weeks after challenge and cultured with PBS or bovine PPD for 20 h. TRIzol homogenates were prepared from these cultures and stored at -80^oC until required for preparation of RNA. An experiment was designed, in consultation with AgResearch Biometricians, to profile gene expression pre-challenge and initially at two of the time points after challenge (5 and 12 weeks). This experiment has used eighteen of the newer microarray slides now available at AgResearch which have 22,000 ESTs. The preparation of labelled RNA and hybridisation of these slides has been completed and analysis of data is currently being conducted.

Complete the study to identify adjuvants and immunostimulants which induce strong cellular immune responses to M. bovis antigens in cattle and evaluate the best combinations of these in vaccines for protection against bovine tuberculosis (funding predominantly from DEFRA, UK).

The adjuvant used with subunit-protein vaccines will be an important factor in whether this type of vaccine can successfully induce protective immune responses to tuberculosis. Unmethylated CG dinucleotides within certain sequence contexts (CpG motifs) have strong immunomodulatory and immunostimulatory capacities. Immune responses to sub-unit protein vaccines may be enhanced by inclusion of specific CpG oligodeoxynucleotides (ODN) to the formulation. A trial in cattle was conducted to find suitable adjuvants for sub-unit protein vaccines which will induce strong protective immune responses to tuberculosis. M. bovis culture filtrate proteins (CFP) was formulated with two different adjuvants, Emulsigen or Polygen, either alone or in combination with a specific ODN (ODN 2007), poly (I:C), a known inducer of IFN-__ or recombinant bovine GM-CSF. Seven different vaccine preparations were tested for their ability to generate humoral and cellular immune responses in cattle. The protective efficacy of two of these CFP/adjuvant combinations that induced the strongest cellular immune responses was determined after experimental challenge of the vaccinated animals with virulent M. bovis.

Vaccines were prepared by mixing M. bovis CFP (0.4 mg per dose) with either Emulsigen or Polygen adjuvant and combining with poly (I:C) (1 mg/dose) or ODN 2007 (250 _g/dose) or recombinant GM-CSF (150 _g/dose). Cattle of 5-6 months age were divided into 7 groups of 5 animals and vaccinated with 2 ml of vaccine subcutaneously. Two further groups of 5 animals were vaccinated subcutaneously with 10⁶ CFU of BCG or were not vaccinated.

All cattle vaccinated with the CFP vaccines were re-vaccinated with the same CFP vaccine at 3 weeks after the initial vaccination. Whole blood was collected at 0, 3, 5.5, 8, 11 and 13 weeks. T-cell responses were measured by the release of IFN-_ and IL-2 from whole blood cultures stimulated with avian and bovine PPD. IFN-_ was measured by ELISA and IL-2 by a bioassay. Serum antibody was measured by ELISA using M. bovis CFP as the antigen. The intradermal tuberculin test was carried out 10 weeks after initial vaccination. Animals were inoculated intradermally with 0.1 mg bovine PPD in the neck and the DTH response measured after 72 hr.

Immunological responses in the animals in the two CFP vaccine groups that showed the strongest IFN-_ responses (CFP/Emulsigen + CpG and CFP/Polygen + CpG) were studied further. The animals in these CFP vaccine groups, together with the BCG-vaccinated and non-vaccinated animals were challenged 13 weeks after the initial vaccination with 5 x 10³ CFU virulent M. bovis. Immune responses were monitored at 2, 5, 9 and 12 weeks after challenge. The animals were killed at 15 weeks after challenge and examined for presence of tuberculous lesions in the lungs and lymph nodes. Samples from the four thoracic lymph nodes (left and right bronchial and anterior and posterior mediastinal) and left and right retropharyngeal lymph nodes were collected from all the animals for bacterial culture and histology.

Humoral responses after vaccination and challenge. Animals vaccinated with the CFP vaccines produced strong antibody responses to M. bovis CFP at 5.5 weeks after initial vaccination. In comparison, weak responses were seen in the BCG group. The highest responses were observed in the groups immunised with vaccines containing CpG ODN. After challenge, antibody responses increased in all the vaccinated groups but then declined. In comparison, antibody responses in the non-vaccinated group progressively increased after challenge.

T cell responses after vaccination and challenge. The BCG-vaccinated animals produced strong IFN-__responses to bovine PPD by 3 weeks after vaccination (Fig.2). Mean responses in the BCG group were higher than mean responses in the non-vaccinated group at 3 weeks and 8-13 weeks after initial vaccination (P<0.01). Weaker IFN-_ responses were observed in the animals vaccinated with the CFP vaccines. However, the mean responses in the groups vaccinated with CFP/Emulsigen + CpG group and CFP/Polygen + CpG were significantly higher than mean responses in the non-vaccinated group. The IFN-_ responses in animals vaccinated with the CFP/Polygen vaccines are shown in Figure 1. At 10 weeks after initial vaccination, none of the CFP-vaccinated cattle gave a DTH response greater than 1.5 mm. In comparison, the BCG-vaccinated animals gave positive skin test responses with a mean response (± SE) of 5.5 ± 0.8 mm.

Pathological findings after challenge with M. bovis. BCG gave a significant reduction in 2 pathological parameters of disease. The median lymph node lesion score (P<0.05) (Fig. 3) and the proportion of thoracic lymph nodes/animal which were M. bovis culture positive (P<0.01) were significantly lower in the BCG group compared to the non-vaccinated group. CFP/Emulsigen + CpG gave a significant reduction in one parameter of disease. The median lymph node lesion score was significantly lower in the CFP/Emulsigen + CpG group compared to the non-vaccinated group (P<0.05) (Fig. 3).

In conclusion, the CFP vaccines induced strong antibody responses but relatively weak IFN-_ responses but the addition of CpG ODN to the vaccines enhanced both antibody and cellular immune responses. The strongest IFN-_ responses to M. bovis CFP vaccines were detected in animals vaccinated with CFP/Emulsigen + CpG ODN and CFP/Polygen + CpG ODN. While BCG gave the best overall protection against tuberculosis, significant protection was also seen in animals vaccinated with the CFP/Emulsigen + CpG vaccine. These results suggest an important role for CpG ODN in stimulating protective Th1 responses to tuberculosis in cattle and CpG ODN should be considered for incorporating into sub-unit protein vaccines.

Development of a serological test to measure immune responses of possums to a possum specific nematode Parastrongyloides trichosuri (collaboration with Mark Ralston).

As work progresses on feasibility studies for the use of Parastrongyloides trichosuri (PT) as a disseminating host for delivery of an agent for the biological management of possums, it becomes important to have a quick reliable method for determining immune responses of possums to the worm. At present this relies on analysis of serum by Western blotting but an ELISA would be cheaper, faster and easier to carry out on large numbers of serum samples. In the current study, antigens that have been trialled in ELISAs include crude homogenised L3 soluble antigens, shown by SDS PAGE to contain the 67kDa antigen used as the diagnostic antigen in Western blots, and further purified fractions of the soluble homogenate prepared by gel filtration. These antigens could distinguish between Western blot negative sera from Stewart Island possums and Western blot positive sera from South Island possums, but could not differentiate between Western blot positive and negative sera from South Island possums. This could due to the fact that PT is not endemic in Stewart Island, neither has it been introduced. South Island possums who are Western blot negative may have been previously exposed to PT and the more sensitive ELISA can detect this. Other antigens are currently being prepared and investigated for their usefulness in an ELISA test.

Immune responses and hormone levels in possums following vaccination with a conjugated GnRH vaccine (collaboration with Doug Eckery).

Vaccines that interfere with gonadotropin (GnRH) activity are candidates for interfering with reproductive capacity as a means of wildlife population control. In white-tailed deer, vaccination with a GnRH vaccine resulted in reduced fawning rates, altered oestrus behaviour, reduced concentrations of progesterone, contraception and failure to maintain pregnancy following conception (Miller et al Am J Reprod. Immunol 2000, 44: 266-74). A GnRH vaccine is a candidate for an agent to control the reproductive capacity of the possum population in New Zealand. The vaccine effect in deer was related to the antibody titre against GnRH. The aim of this work was to use a conjugated GnRH vaccine similar to the one used in deer to determine whether immune responses could be detected in possums and to follow hormone levels to establish whether vaccination altered hormones important for reproductive function in the possum.

Male possums that had been castrated were used as any changes in hormone levels are more marked in these animals. One group of possums (n=6) was vaccinated with GnRH complexed to keyhole limpet haemacyanin (KLH) formulated in a oil based adjuvant kindly supplied by a collaborator (L. Miller, National Wildlife Centre, Fort Collins, USA). The other group was vaccinated with KLH emulsified in Freund's adjuvant to act as controls (n=6). Possums received a booster vaccination 5 weeks after the first vaccination. Blood samples were collected over an 11 week period for determination of cell-mediated and antibody responses and levels of luteinising hormone (LH) and follicle stimulating hormone (FSH).

Antibody responses to GnRH were detected in 5/6 GnRH vaccinated possums (animal numbers 854, 858, 863, 878, 6382) and of those five, three gave a slightly stronger response (854, 858, 6382). Antibodies to GnRH were undetectable in control possums immunised with KLH alone (Fig. 4) but KLH specific antibodies were detected (Fig. 4 and 5). GnRH activity was neutralised in 3/6 GnRH vaccinated possums (animal numbers 854, 858, 6382) as evidenced by a decline in luteinizing hormone (LH) concentrations following the booster vaccination. Again no effect was seen in the controls (Fig. 6). Follicle stimulating hormone (FSH) concentrations were also affected in the same GnRH vaccinated possums, but not to the same extent as for LH (Fig. 7). This is not entirely surprising since FSH is known to be regulated by other factors in addition to GnRH. Thus, the hormone data was in agreement with the antibody responses. The three possums with the highest antibody response were also the ones whose hormone levels were affected by the treatment. The low and non-specific cell-mediated immune responses to GnRH were not reflected in antibody responses or hormone levels. Immunisation with a gonadotrophin (GnRH) vaccine is being investigated as a possible means of possum population control. These results, of the first study of GnRH vaccination of possums, give promising preliminary data, which may be improved by increasing the dosage of the GnRH vaccine or giving another booster vaccination.

Immunisation of possums with a zona pellucida antigen and BCG for a dual purpose vaccine (collaboration with Janine Duckworth, LandCare)

Possums have previously been immunised intranasally with keyhole limpet haemacyanin (KLH) mixed with BCG to test the feasibility of using BCG not only as a vaccine for tuberculosis in possums but also as a mucosal adjuvant for the delivery of an immunocontraceptive or immunosterilisation agent. As it was demonstrated that BCG can act as a mucosal adjuvant in possums using a model antigen, an experiment was carried out to test whether recombinant possum zona pellucida antigen 3 (ZP3), a candidate immunocontraceptive agent, can be adjuvanted with BCG for mucosal delivery via the nose. Groups of female possums (n= 7) were immunised intranasally/conjunctivally with either 125 _g ZP3, 10⁶ CFU BCG, 125 _g ZP3 plus 10⁶ CFU BCG, or subcutaneously with 60 _g ZP3 in Freund's complete adjuvant. After 3 weeks they were re-vaccinated in the same way except Freund's incomplete adjuvant was used for subcutaneous vaccination. Blood was collected at 0, 4, and 9 weeks after initial vaccination for measuring ZP3 specific antibody in the serum and lymphocyte proliferative assay (LPA) responses to PPD and ZP3. At 9 weeks the possums were sacrificed and washings from the vagina, uterus and oviduct were collected together with follicular fluid and ZP3 specific antibody was measured by ELISA.

LPA responses to ZP3 were optimised with respect to dose of ZP3 in the assay. The strongest responses were obtained at sacrifice (9 weeks) from 3 animals vaccinated with ZP3 plus Freund's adjuvant given subcutaneously using 10 _g/ml ZP3 in vitro and are shown in Fig. 8. These three possums also had the highest serum ZP3 specific antibody titres at this time. Thus the LPA data appeared to correlate with ZP3 specific serum antibody. Although one of the possums (# 922) had detectable ZP3 specific antibody (OD 0.22) in vaginal secretions (OD 0.23) and follicular fluid (OD 0.77), another had detectable antibody only in follicular fluid (OD 0.30) and other possums which did not exhibit LPA responses had detectable antibody in mucosal secretions.

When ZP3 was administered via mucosal routes with BCG, LPA responses to ZP3 were not detected in any possums, neither was ZP3 specific serum antibody but antibody was detected in follicular fluid of 4/6 possums (OD 0.22-0.40) and in vaginal washings of 3/6 possums (OD 0.25-0.43) (Figs. 9 and 10). Thus the LPA did not appear to correlate with antibody at reproductive sites. Vaccination with ZP3 alone or BCG alone resulted in one possum from each group having detectable antibody in the follicular fluid but not in samples from the other reproductive sites.

As in serum antibody responses in possums vaccinated with ZP3 in Freund's was lower than in previous experiments (J Duckworth personal communication) it is possible that the ZP3 preparation used in this experiment was not as immunogenic as that used in previous work. However, the production of ZP3 specific antibody at reproductive sites after mucosal immunisation via the nose with BCG plus ZP3 are encouraging and warrant repeating the experiment using oral delivery.

Publications

Publications (July 2002- June 2003)

Publications in peer-reviewed scientific journals

Skinner, M.A., Parlane,N., McCarthy, A., Buddle, B.M. 2003. Cytotoxic T cell responses to Mycobacterium bovis during experimental infection of cattle with bovine tuberculosis Immunology (submitted).
Doolin, E.E., Buddle, B.M., McLeod, B., Mahoney, T., Skinner, M.A. 2003. Mucosal immunity in the brushtail possum (Trichosurus vulpecula). Detection of antibody at male and female reproductive sites. Biological Procedures Online (submitted).
Buddle, B.M., Wedlock, D.N., Parlane, N.A., Corner, L.A.L., de Lisle, G.W. and Skinner, M.A. 2003. Revaccination of neonatal calves with Mycobacterium bovis BCG reduced the level of protection against tuberculosis induced by a single vaccination. Infect. Immun. (submitted).
Skinner, M.A., Buddle, B.M., Wedlock, D.N., Keen, D.L., de Lisle, G.W., Tascon, R.E., Ferraz, J.C., Lowrie, D.B., Cockle, P.J., Vorderemier, H.M. and Hewinson, R.G. 2003. A DNA prime- BCG boost vaccination strategy in cattle induces protection against bovine tuberculosis. Infect. Immun. (submitted).
Aldwell, F.E., Keen, D., Parlane, N., Skinner, M.A., de Lisle, G.W. and Buddle, B.M. 2003. Oral vaccination with Mycobacterium bovis BCG in a lipid formulation induces resistance to pulmonary tuberculosis in possums. Vaccine (submitted).
Wedlock, D.N., Skinner, M.A., Parlane, N.A., Vordermeier, H.M., Hewinson, R.G., deLisle, G.W., Buddle, B.M. 2003. Vaccination with DNA vaccines encoding MPB70 or MPB83 or a MPB70 prime/protein boost does not protect cattle against bovine tuberculosis. Tuberculosis (in press).
Buddle, B.M., McCarthy, A.R., Ryan, T.J., Pollock, J.M., Vordermeier, R.G., Hewinson, R.G., Andersen, P., and de Lisle, G.W. 2003. Use of mycobacterial peptides and recombinant proteins for the diagnosis of bovine tuberculosis in skin test-positive cattle. Vet Rec. (in press).
Corner, L.A., Buddle, B.M. and Morris, R.S. 2003. Infection of the brushtail possum (Trichosurus vulpecula) with bovine tuberculosis by the conjunctival route. Vet. J. (in press).
Buddle, B.M. Pollock, J.M., Skinner, M.A. and Wedlock, D.N. 2003. Development of vaccines to control bovine tuberculosis in cattle and relationship to vaccine development for other intracellular pathogens. Int. J. Parasitol. 33: 555 - 566.
Skinner, M.A., Ramsay, A., Buchan, G.S., Keen, D.L., Ranasinghe, C., Slobbe, L., Collins, D.M., de Lisle, G.W. and Buddle, B.M 2003. A DNA prime-live vaccine boost strategy in mice can augment IFN-_ responses to mycobacterial antigens but does not increase the protective efficacy of two attenuated strains of Mycobacterium bovis against bovine tuberculosis. Immunology 108: 548-555.
Aldwell, F.E., Tucker, I.G., de Lisle, G.W. and Buddle, B.M. 2003. Oral delivery of Mycobacterium bovis BCG in a lipid formulation induces resistance to pulmonary tuberculosis in mice. Infect Immun. 71: 101-108.
Hewinson, R.G., Vordermeier, H.M., Buddle, B.M. 2003. Use of the bovine model of tuberculosis for the development of improved vaccines and diagnostics. Tuberculosis 83: 119-130.
Doolin, E.E., Wedlock, D.N., Buddle, B.M. and Skinner, M.A. 2002. Mucosal immunity in the brushtail possum (Trichosurus vulpecula). Detection of antibody in serum and at female reproductive sites after intranasal immunisation. Immunol. Cell. Biol. 80: 358-363.
Cockle, P.J., Gordon, S.V., Lalvani, A., Buddle, B.M., Hewinson, R.G. and Vordermeier, H.M. 2002. Identification of novel Mycobacterium tuberculosis antigens with potential as diagnostic reagents or sub-unit vaccine candidates by comparative genomics. Infect. Immun. 70: 6996-7003.
Collins, D.M., Wilson, T., Campbell, S., Buddle, B.M., Wards, B.J., Hotter, G. and de Lisle, G.W. 2002. Production of avirulent mutants of Mycobacterium bovis with vaccine properties by the use of illegitimate recombination and screening of stationary phase cultures. Microbiology 148: 3019-3027.
Corner, L.A.L., Pfeiffer, D.U., de Lisle, G.W., Morris, R.S. and Buddle, B.M. 2002. Natural transmission of Mycobacterium bovis infection in captive brushtail possums (Trichosurus vulpecula). N.Z. Vet. J. 50: 154-162.
Corner, L.A., Norton, S., Buddle, B.M. and Morris, R.S. 2002. The efficacy of bacilli Calmette-Guerin vaccine in wild brushtail possums (Trichosurus vulpecula) Res. Vet. Sci. 73, 145-152.
Buddle, B.M., Skinner, M.A., Wedlock, D.N., Collins, D.M. and de Lisle, G.W. 2002. New generation vaccines and delivery systems for control of bovine tuberculosis in cattle and wildlife. Vet. Immunol. Immunopathol. 87: 177-185. .
Young, S., O'Donnell, M., Lockhart, E., Buddle, B., Slobbe, L., Lyo, Yi and Buchan G. 2002. Manipulation of immune responses to Mycobacterium bovis by vaccination with IL-2 and IL-18 secreting recombinant bacillus Calmette Guerin. Immunol. Cell Biol. 80: 209-215.

Book chapter

Buddle, B.M., Pollock, J.M. and Hewinson, R.G. 2003. Experimental infection models of tuberculosis in domestic and wild animals. In: Cole, S., Eisenach, Gicquei, B., McMurray, D.M., Jacobs, W., eds. Tuberculosis. Washington, DC: American Society of Microbiology (submitted).

Conference papers and seminars

Buddle, B.M. 2002. Research from the Infectious Diseases Group AgResearch Wallaceville, Immunet meeting, Wellington, 4 July 2002.
Skinner, M.A. 2002. New vaccine strategies for bovine tuberculosis: the kiwi experience. Seminar. Veterinary Laboratory Agency, Weybridge, UK, July 2002.
Skinner, M.A. 2002. New vaccines for tuberculosis. Seminar. School of Biological Science Victoria University, Wellington, Oct 2002.
Parlane, N. 2002. Seminar, Department of Agriculture for Northern Ireland, Belfast, UK. Oct. 2002.
Buddle, B.M. 2002. Bovine tuberculosis: improved diagnostic tests, oral vaccines for wildlife and neonatal calf vaccination. Seminars, 1) DEFRA, London, UK, 13 Nov. 2002; 2) VLA Weybridge, UK 15 Nov. 2002; 3) University College Dublin, Dublin, Ireland, 18 Nov. 2002; 4) DARDNI, Belfast UK 20 Nov. 2002.
Wedlock, D.N., Skinner, M.A., Buddle, B.M. 2002. Improving the efficiency of BCG vaccination for neonates. Australasian Society for Immunology Conference, Brisbane, Australia, 9-13 Dec. 2002.
Skinner, M.A. 2002. Prime boost vaccination strategies for tuberculosis. Seminar. Gene Therapy Program, Louisiana State University, New Orleans, USA, Dec 2002.
Skinner, M.A. 2002. Improving the efficiency of BCG vaccination for neonates. Sequella Global Tuberculosis Foundation, Vaccine 2002 Conference, Washington, DC, USA, Dec. 2002.
Buddle, B.M., Skinner, M.A., Wedlock, M.A. 2003. Reduction in the efficacy of BCG vaccine for protection against bovine tuberculosis with two vaccinations of BCG in neonatal calves. Keystone symposium, "Tuberculosis: Integrating host and pathogen biology", Taos, New Mexico, USA, 25-30 Jan 2003.
Cockle, P.J., Gordon, S.V., Lalvani, A., Buddle, B.M., Hewinson, R.G., Vordermeier, H.M. 2003. Indentification of novel Mycobacterium tuberculosis antigens with potential as diagnostic reagents or sub-unit vaccine candidates by comparative genomics. Keystone symposium, "Tuberculosis: Integrating host and pathogen biology", Taos, New Mexico, USA, 25-30 Jan 2003.
Buddle, B.M. 2003. Cattle as a model for human tuberculosis. Seminar, Colorado State University, Fort Collins, USA, 31 January 2003.
Skinner, M.A., 2003. GE issues. Auckland College of Education students. April 2003.
Buddle, B.M. 2003. Diagnostic tests for control of bovine tuberculosis. Seminar. University College Dublin, Dublin, Ireland, 15 May 2003.
Wedlock, D.N., Skinner, M.A., Vordermeier, H.M., Hewinson, R.G., Hecker, R., van Drunen Little-van den Hurk, S., Buddle, B.M. 2003. Addition of CpG ODN to a sub-unit protein vaccine enhances immune responses and protection against tuberculosis in cattle. Conference - Modern Vaccines Adjuvants and Delivery Systems. Dublin, Ireland, 4-6 June 2003.
Wedlock, D.N. 2003. Development of tuberculosis vaccines for cattle and possums. Seminar, Institute for Animal Health, Compton, UK, 9 June 2003.
Wedlock, D.N. 2003. Development of tuberculosis vaccines for cattle and possums. Seminar, Veterinary Laboratories Agency, Weybridge, UK, 10 June 2003.

Contact for Enquiries

Farm Monitoring Programme Manager
Monitoring and Evaluation
MAF Policy
PO Box 2526
Wellington
NEW ZEALAND
Phone: +64 4 894 0623
Fax: +64 4 894 0741
Contact this person

5. Possum Biocontrol Category

5.1 PBC 251

Blocking embryonic development in Brushtail Possums

OBJECTIVE 1: APPROACH

Alternative expression systems tested for VAP5 protein expression.

OBJECTIVE 1, OUTCOMES:

VAP5 protein production

Objective 2: APPROACH

Protein production - CP4

Objective 2: OUTCOMES

Protein production - CP4

Objective 3: OUTCOMES

AIM:

CONTEXT:

APPROACH

OUTCOMES

CONTEXT

APPROACH

OUTCOMES

HIGHLIGHTS

PUBLICATIONS

5.2 PBC 252

Possum viruses

Summary

Background

Approach & Outcomes

Publications

5.3 PBC 254

Oocyte growth factors

Summary

Objective 1 - Immunisation of possums against GDF-9 and GDF-9B

Background

Approach & Outcomes

5.4 PBC 255

Controlled oestrus

Summary

References

Background

Approach & Outcomes

Objective 2

5.5 PBC 256

Oral bioactives

Summary

Background

Annual Report 02-03

Objective 1: Formulation for delivery to hindgut

Approach & Outcomes

Publications

5.6 PBC 257

Transgenic worms

Summary

Background

Approach & Outcomes

5.7 PBC 258

Bovine Tb

Summary

Background

b) Possum Immunology

Description

Summary

Approach & Outcomes

Field trial to determine the efficacy of an oral bait BCG for wild possums.

Publications

Contact for Enquiries

Search Rural NZ

Keyword Search