Peptide Presentations of Marsupial MHC Class I Visualize Immune Features of Lower Mammals Paralleled with Bats.

Marsupials are one of three major mammalian lineages that include the placental eutherians and the egg-laying monotremes. The marsupial brushtail possum is an important protected species in the Australian forest ecosystem. Molecules encoded by the MHC genes are essential mediators of adaptive immune responses in virus-host interactions. Yet, nothing is known about the peptide presentation features of any marsupial MHC class I (MHC I). This study identified a series of possum MHC I Trvu-UB*01:01 binding peptides derived from wobbly possum disease virus (WPDV), a lethal virus of both captive and feral possum populations, and unveiled the structure of marsupial peptide/MHC I complex. Notably, we found the two brushtail possum-specific insertions, the 3-aa Ile52Glu53Arg54 and 1-aa Arg154 insertions are located in the Trvu-UB*01:01 peptide binding groove (PBG). The 3-aa insertion plays a pivotal role in maintaining the stability of the N terminus of Trvu-UB*01:01 PBG. This aspect of marsupial PBG is unexpectedly similar to the bat MHC I Ptal-N*01:01 and is shared with lower vertebrates from elasmobranch to monotreme, indicating an evolution hotspot that may have emerged from the pathogen-host interactions. Residue Arg154 insertion, located in the α2 helix, is available for TCR recognition, and it has a particular influence on promoting the anchoring of peptide WPDV-12. These findings add significantly to our understanding of adaptive immunity in marsupials and its evolution in vertebrates. Our findings have the potential to impact the conservation of the protected species brushtail possum and other marsupial species.

Serological evidence for the presence of wobbly possum disease virus in Australia.

Wobbly possum disease virus (WPDV) is an arterivirus that was originally identified in common brushtail possums (Trichosurus vulpecula) in New Zealand, where it causes severe neurological disease. In this study, serum samples (n = 188) from Australian common brushtail, mountain brushtail (Trichosurus cunninghami) and common ringtail (Pseudocheirus peregrinus) possums were tested for antibodies to WPDV using ELISA. Antibodies to WPDV were detected in possums from all three species that were sampled in the states of Victoria and South Australia. Overall, 16% (30/188; 95% CI 11.0-22.0) of possums were seropositive for WPDV and 11.7% (22/188; 95% CI 7.5-17.2) were equivocal. The frequency of WPDV antibody detection was the highest in possums from the two brushtail species. This is the first reported serological evidence of infection with WPDV, or an antigenically similar virus, in Australian possums, and the first study to find antibodies in species other than common brushtail possums. Attempts to detect viral RNA in spleens by PCR were unsuccessful. Further research is needed to characterise the virus in Australian possums and to determine its impact on the ecology of Australian marsupials.

Characterization of antibody V segment diversity in the Tasmanian devil (Sarcophilus harrisii).

The Tasmanian devil (Sarcophilus harrisii) immune system has recently been under scrutiny because of the emergence of a contagious cancer, which has decimated devil numbers. Here we provide a comprehensive description of the Tasmanian devil immunoglobulin variable regions. We show that heavy chain variable (VH) and light chain variable (VL) repertoires are similar to those described in other marsupial taxa: VL diversity is high, but VH diversity is restricted and belongs only to clan III. As in other mammals, one VH and one Vλ germline family and multiple incomplete Vκ germline sequences were identified in the genome. High Vκ variation was observed in transcripts and we predict that it may have arisen by gene conversion and/or somatic mutations, as it does not appear to have originated from germline variation. Phylogenetic analyses revealed that devil VL gene segments are highly complex and ancient, with some lineages predating the separation of marsupials and eutherians. These results indicate that although the evolutionary history of immune genes lead to the expansions and contractions of immune gene families between different mammalian lineages, some of the ancestral immune gene variants are still maintained in extant species. A high degree of similarity was found between devil and other marsupial VH segments, demonstrating that they originated from a common clade of closely related sequences. The VL families had a higher variation than VH both between and within species. We suggest that, similar to other studied marsupial species, the complex VL segment repertoire compensates for the limited VH diversity in Tasmanian devils.

Sustained protection against tuberculosis conferred to a wildlife host by single dose oral vaccination.

BACKGROUND: Vaccination of wildlife against bovine tuberculosis (TB) is being considered by several countries to reduce the transmission of Mycobacterium bovis infection to livestock. In New Zealand, where introduced brushtail possums (Trichosurus vulpecula) are the major wildlife hosts, we have previously shown that repeat applications of a lipid-encapsulated oral bacille Calmette-Guerin (BCG) vaccine reduce the incidence of naturally acquired TB in wild possums. Here we extend this conceptual demonstration to an operational level, assessing long-term protection against TB conferred to free-living possums by a single oral immunisation. METHODS: Possums in a non-TB area were randomly allocated to receive lipid-formulated BCG vaccine or remained unvaccinated. After initial trials to assess vaccine immunogenicity and establishment of protection within the first year post-vaccination, 13 individuals of each treatment group were relocated to a biosecurity facility and challenged (at 28 months post-vaccination) by subcutaneous injection of virulent M. bovis. RESULTS: Vaccine immunogenicity and short-term protection were confirmed at 2 months and 12 months post-vaccination, respectively. In the long-term assessment, vaccinated possums had significantly reduced bacterial counts in peripheral lymph nodes compared to controls, with 0.6-2.3 log(10)-fold reductions in M. bovis burdens. DISCUSSION: The magnitude of protective response by possums to experimental challenge at 28 months post-vaccination is known to equate to a high degree of protection against natural infection in this species. With techniques for oral bait delivery well advanced, the longevity of protection demonstrated here shows that an operable wildlife vaccine against TB is feasible.

Humoral immune responses in brushtail possums (Trichosurus vulpecula) induced by bacterial ghosts expressing possum zona pellucida 3 protein.

The introduced common brushtail possum (Trichosurus vulpecula) is a major pest in New Zealand and immunocontraceptive vaccines are being developed for biocontrol of possum populations, with bacterial ghosts (BGs) being evaluated as a means of oral delivery. Recombinant BGs expressing possum zona pellucida 3 protein (ZP3) as an L' membrane-anchored protein (ZP3-L') or as an S-layer SbsA-fusion protein (MBP-SbsA-ZP3) were produced by the expression of the cloned bacteriophage phiX174 lysis gene E in E. coli NM522. The humoral immune responses of possums immunised with BGs expressing possum ZP3 were investigated following oral, intranasal/conjunctival, parenteral, and intraduodenal administration to evaluate the BG-ZP3 system for possum fertility control. Antibodies to possum ZP3 were detected in the serum, oviduct secretions, and follicular fluid of immunised animals. Intranasal/conjunctival immunisation elicited reliable antibody immune response in serum and at a key effector site, the ovarian follicular fluid. Intraduodenal administration of possum ZP3 BG vaccine as a priming immunisation elicited significant systemic immune responses, but oral immunisation did not, indicating that protection of BG vaccines from degradation by gastric acidity would enhance the effectiveness of orally delivered vaccines. The detection of antibodies at elevated levels at target sites in the reproductive tract following mucosal delivery demonstrates, for the first time, the potential of BGs as an effective system for vaccine delivery to wild animals, and intranasal/conjunctival immunisation as a promising means for delivery of immunocontraceptive vaccines to wild animals.

Identification and evaluation of an infertility-associated ZP3 epitope from the marsupial brushtail possum (Trichosurus vulpecula).

Immunologically based fertility control vaccines against zona pellucida (ZP) proteins are being developed in New Zealand for biocontrol of the brushtail possum (Trichosurus vulpecula), an introduced Australian marsupial pest. We have shown that immunization of female possums with recombinant possum ZP3 protein (rZP3) reduced fertility by 79%. To enhance the specificity of possum immunocontraceptive vaccines, B-cell epitopes on possum ZP3 protein were mapped using sera of female possums immunized with possum rZP3 and subjected to a fertility trial. The amino acid sequence of the full-length possum ZP3 protein was used to synthesize a complete set of 83 (12-mer) biotinylated peptides each with an overlap of five amino acids with the neighboring peptides. The peptides were used in a modified enzyme-linked immunosorbent assay (ELISA) to identify continuous epitopes recognized by antibodies in the sera of possums immunized with possum rZP3. Sixteen epitopes were identified on the possum ZP3 protein. Comparison of the ELISA binding patterns of these peptides to antibodies in the individual sera with the fertility status of rZP3-immunized possums identified only one epitope (amino acids 156-172) to be associated with infertility. However, female possums immunized with this epitope showed no significant reduction in fertility. The possible reasons for the failure of this potential infertility epitope are discussed.

Bacterial ghosts as a delivery system for zona pellucida-2 fertility control vaccines for brushtail possums (Trichosurus vulpecula).

The introduced brushtail possum is a serious pest in New Zealand and there is much interest in the development of an immunocontraceptive vaccine for population control. Immunisation of female possums against recombinant possum zona pellucida protein-2 (ZP2) is known to reduce embryo production by 72-75% but successful development of fertility control will depend on a delivery system that is effective for field use. Bacterial ghost vaccine technology is a promising system to formulate a non-living vaccine for bait or aerosol delivery. The N-terminal (amino acid residues 41-316, ZP2N) and C-terminal (amino acid residues 308-636, ZP2C) regions of possum ZP2 were fused to maltose-binding protein and expressed in the periplasmic space of Escherichia coli NM522 bacterial ghosts. Female possums (n=20 per treatment group) were immunised with 20mg of either plain ghosts, ZP2N ghosts, or ZP2C ghosts in phosphate-buffered saline applied to the nostrils and eyes (nasal/conjunctival mucosa) at weeks 0, 2 and 4. Effects of immunisation on fertility were assessed following superovulation and artificial insemination. Both constructs evoked humoral (antibody) and cell-mediated immune responses in possums and significantly fewer eggs were fertilised in females immunised against ZP2C ghosts. Results in this study indicate that bacterial ghosts containing possum ZP antigens can reduce possum fertility when delivered by mucosal immunisation and offer a promising delivery system for fertility control of wild possum populations.

High variability in the MHC class II DA beta chain of the brushtail possum (Trichosurus vulpecula).

The diversity of class II major histocompatibility complex (MHC) loci was investigated in the brushtail possum, an important marsupial pest species in New Zealand. Immunocontraception, a form of fertility control that generates an autoimmune response, is being developed as a population control method for the possum. Because the immune response is partly under genetic control, an understanding of immunogenetics in possum will be crucial to the development of immunocontraceptive vaccines. MHC molecules are critical in the vertebrate immune response. Class II MHC molecules bind and present exogenously derived peptides to T lymphocytes and may be important in the presentation of immunocontraceptives. We used polymerase chain reaction primers designed to amplify the peptide binding region of possum class II MHC genes to isolate sequences from 49 animals. We have previously described 19 novel alleles from the DAB locus in the possum (Holland et al., Immunogenetics 60:449-460, 2008). Here, we report on another 11 novel alleles isolated from possum DAB, making this the most diverse marsupial locus described so far. This high level of diversity indicates that DAB is an important MHC locus in the possum and will need to be taken into account in the design of immunocontraceptive vaccines.

Vesicle-associated protein 1: a novel ovarian immunocontraceptive target in the common brushtail possum, Trichosurus vulpecula.

Ovarian-based immunological research is currently restricted to proteins of the zona pellucida. This study examined the immunocontraceptive potential of a novel vesicle-associated protein, VAP1, previously isolated from the vesicle-rich hemisphere of the brushtail possum oocyte. Seven female possums were immunized against recombinant glutathione S-transferase-VAP1 fusion protein. Control animals (n=3) received antigen-free vaccinations. Following immunization, regular blood sampling determined the level and duration of immune response. Animals were monitored daily, pre- and post-immunization, to determine estrous cycling activity and the percentage of reproductive cycles yielding viable young. The reproductive tracts and somatic organs of VAP1-immunized (n=7), control-immunized (n=3) and non-immunized (n=5) animals were collected and examined by histology and transmission electron microscopy. VAP1 immunization caused a strong and sustained immune response. Elevated levels of VAP1 antibody binding were detected in sera following initial injections, and immune titers rose as boosters were administered. Immunization had no adverse effect upon animal behavior or body condition. Immunized females demonstrated no major change in annual estrous cycling activity; however, the percentage of reproductive cycles resulting in pouch young decreased significantly (P<0.05) by 40%. Histological and ultrastructural analyses revealed an abundance of lipid-like degradation bodies within the ooplasm of developing oocytes and the cytoplasm of failing uterine zygotes. Active macrophage invasion of enlarged endometrial glands was observed in the uteri of two females. Reproductive tract changes are discussed in relation to observed fertility decline. The results of this study indicate that VAP1 has exciting potential as an immunocontraceptive target for possum control in New Zealand.

Novel alleles in classical major histocompatibility complex class II loci of the brushtail possum (Trichosurus vulpecula).

We have investigated the diversity of class II major histocompatibility complex (MHC) loci in the brushtail possum (Trichosurus vulpecula), an important marsupial pest species in New Zealand. Immunocontraceptive vaccines, a method of fertility control that employs the immune system to attack reproductive cells or proteins, are currently being researched as a means of population control for the possum. Variation has been observed in the immune response of individual possums to immunocontraceptives. If this variability is under genetic control, it could compromise vaccine efficacy through preferential selection of animals that fail to mount a significant immune response and remain fertile. The MHC is an important immune region for antigen presentation and as such may influence the response to immunocontraceptives. We used known marsupial MHC sequences to design polymerase chain reaction primers to screen for possum MHC loci. Alpha and beta chains from two class II families, DA and DB, were found in possums throughout New Zealand. Forty new class II MHC alleles were identified in the possum, and the levels of variability in the MHC of this marsupial appear to be comparable to those of eutherian species. Preliminary population surveys showed evidence of clustering/variability in the distribution of MHC alleles in geographically separate locations. The extensive variation demonstrated in possums reinforces the need for further research to assess the risk that such MHC variation poses for long-term immunocontraceptive vaccine efficacy.

Oral challenge of brushtail possums (Trichosurus vulpecula) with possum enteroviruses: clinical observation, antibody response and virus excretion in faeces.

AIM: To investigate the pathogenesis of two cytopathic enteroviruses, W1 and W6, isolated from possums, to evaluate their potential as vectors for biological management of possums. METHODS: Possums (n=10) were fed 1 x 107 median tissue culture infectious doses (TCID50) of either the W1 strain (Possums 1-5) or W6 strain (Possums 6-10), while controls (n=2, Possums 11 and 12) were fed uninfected cell culture medium. Blood samples were collected from all possums on Days -1, 2, 7, 14, 21 and 34 or 35 post-inoculation (p.i.), and bodyweight and rectal temperatures were measured on the same days. Virus specific antibodies were determined using virus neutralisation tests (VNTs). Faeces were collected on Days -1, 2, 7, 9, 14, 16, 21, 28 and 34 or 35 p.i., and the presence of W1 and W6 virus in faeces was determined using cell culture and reverse transcriptase-polymerase chain reaction (RT-PCR). Possums were euthanised on Day 34 or 35, and ileal Peyer's patches were collected for detection of virus using RT-PCR. RESULTS: No diarrhoea or significant changes in rectal temperature or loss of bodyweight were observed in virus-inoculated possums during the study period. Virus-neutralising antibodies were detected using VNTs in 2/5 and 3/5 possums exposed to the W1 and W6 strain, respectively. Excretion of virus in faeces was detected in 6/10 virus-inoculated possums, from as early as Day 4 p.i.; virus excretion in faeces was transient in some and persistent in other possums up to the time of euthanasia. The viruses were detected in 3/10 virus-inoculated possums following necropsy, but were not detected using RT-PCR in sera collected on Days 2 and 7, nor in Peyer's patches of virus-inoculated possums collected on Day 34 or 35. CONCLUSIONS: Possum enteroviruses W1 and W6 established an asymptomatic infection in the gastrointestinal tract (GIT) in a proportion of challenged possums. A virus-specific antibody response was elicited in infected possums, which excreted the virus in faeces for up to 35 days p.i. The absence of noticeable adverse effects in enterovirus-infected possums is an advantageous characteristic for candidate vectors on animal welfare grounds.

A new attenuated Mycobacterium bovis vaccine protects brushtail possums (Trichosurus vulpecula) against experimental tuberculosis infection.

Vaccination of wildlife against bovine tuberculosis is being actively considered in countries that have wildlife reservoirs of Mycobacterium bovis infection. A newly attenuated strain of M. bovis (WAg533) was produced as part of a programme to develop a better vaccine than BCG to control tuberculosis in brushtail possums in New Zealand. The vaccine efficacy of WAg533 in possums was compared to BCG using three different methods of inoculation (conjunctival/intranasal, oral and sub-cutaneous) followed by aerosol challenge. Overall, WAg533 was a more potent vaccine than BCG and by two methods of inoculation gave more measures of protection that were significantly different from controls.

Oral vaccination of brushtail possums (Tichosurus vulpecula) with BCG: immune responses, persistence of BCG in lymphoid organs and excretion in faeces.

AIMS: To determine immune responses, and the localisation and persistence of Mycobacterium bovis bacille Calmette-Guérin (BCG) in gut-associated lymphoid tissues (GALT) and other organs in possums vaccinated orally with lipid-formulated BCG vaccine. To determine the duration of excretion and longevity of survival of BCG in the faeces of vaccinated animals. METHODS: Possums (n=28) were vaccinated with lipid-formulated BCG (1 x 10(8) colony forming units (cfu) of formulated BCG) by the oral route. Control possums (n=17) were fed oral bait pellets containing formulation medium only. Possums were sacrificed at 3 days and at 1, 3, 6 and 8 weeks after vaccination or ingestion of bait. Proliferation responses to bovine purified protein derivative (PPD) were measured in lymphocytes from blood and mesenteric lymph nodes (MLN) and samples of lung, spleen, liver, MLN and Peyer's patches (PP) were cultured for the presence of BCG. The number of BCG organisms excreted in faeces and the duration of excretion were determined in eight vaccinated possums and eight control possums over a 3-week period. In a separate experiment, a further six possums were vaccinated with oral BCG vaccine (5-10 x 10(8) cfu BCG/possum) and their faeces collected over 48-72 h, for culture of BCG. The longevity of survival of BCG in these faeces was determined by storing faecal samples (n=12) under three different conditions: in an incubator (22.5 degrees C), and conditions which simulated the forest floor and open pasture. A proportion (1-2 g) of these faecal samples was collected after storage for 1, 3, 5, 8 or 20 weeks, and cultured for BCG. RESULTS: Possums vaccinated orally with BCG vaccine showed strong proliferation responses to bovine PPD in peripheral blood lymphocytes at 6-8 weeks post-vaccination (p.v.). Positive lymphocyte proliferation assay (LPA) responses to bovine PPD were first evident in MLN at 3 weeks p.v. BCG was cultured from MLN and PP in a proportion of animals at 3-8 weeks p.v. BCG was not cultured from sections of spleen, lung or liver at any time p.v. BCG was recovered in low to moderate numbers from the faeces of vaccinated possums for up to 7 days, and maximal numbers were cultured in faeces collected 48-72 h p.v. After storage for 1 week, BCG was cultured from all faecal samples placed in the incubator and from a proportion of faeces exposed to conditions similar to those on the forest floor and pasture. With the exception of one faecal sample stored under forest floor conditions which was culture-positive for BCG at 3 and 5 weeks, BCG was not cultured from any other faecal sample stored for more than 1 week. CONCLUSIONS: Ingestion of oral BCG vaccine by possums was associated with the development of strong cell-mediated immunity in both blood and MLN. Following oral vaccination with BCG, the organisms were localised and persisted in GALT but did not spread to the spleen, liver or lungs. BCG was shed in low to moderate numbers in the faeces for up to 7 days p.v. The viability of BCG excreted in faeces decreased rapidly, particularly when faeces were exposed to an open pasture environment. Oral vaccination of possums with formulated BCG is unlikely to result in undue contamination of the environment with BCG.

Susceptibility of brushtail possums (Trichosurus vulpecula) infected with Mycobacterium bovis is associated with a transient macrophage activation profile.

The Australian brushtail possums are highly susceptible to Mycobacterium bovis and are the principal wildlife reservoir of M. bovis in New Zealand. To better understand the disease process in these animals, brushtail possums were infected by the aerosol route with a virulent strain of M. bovis, and immune parameters measured. M. bovis replicated actively in the lungs of infected animals. Animals began developing macroscopic lung lesions at 4 and 5 weeks following infection, with some lesions appearing in the livers and spleens. Infection determined the emergence of blood lymphocytes which proliferated in response to bovine purified protein derivative from M. bovis (PPD-b) at 3, 4 and 5 weeks. The response to a mitogen (Concanavalin A) waned progressively with time. Infection was associated with a modest increase in the numbers of free lung cells. Nitrite was detectable in the lavage fluids of infected animals at 3 weeks postinfection, but not at 4 and 5 weeks. Macrophage activation in the lungs was evident as alveolar macrophages produced more oxidants, significant levels of nitric oxide (NO), as well as tumor necrosis factor alpha (TNF-alpha) bioactivity at 3 weeks postinfection. However, macrophages from infected animals lost the ability to generate nitrite- and TNF-alpha generation was depressed at 4 and 5 weeks postinfection, the time at which macroscopic lesions in the lungs became apparent. Alveolar macrophages from animals at 3 weeks postinfection blocked the replication of M. bovis in part via a NO-dependent mechanism, and were more refractory for M. bovis growth than cells from naïve animals to bacterial replication. Alveolar macrophages from animals at 4 and 5 weeks postinfection allowed substantial replication of M. bovis, and no NO-dependent bacteriostatic activity was apparent. Introduction of autologous lymphocytes from the blood of infected animals in co-cultures rendered infected macrophages more resistant to M. bovis replication. We conclude that M. bovis infection in brushtail possums is associated with a transient activation of alveolar macrophages, although in vitro exposure to sensitized T cells can enhance this profile.

Unusually similar patterns of antibody V segment diversity in distantly related marsupials.

A pattern of coevolution between the V gene segments of Ig H and L chains has been noted previously by several investigators. Species with restricted germline V(H) diversity tend to have limited germline V(L) diversity, whereas species with high levels of germline V(H) diversity have more diverse V(L) gene segments. Evidence for a limited pool of V(H) but diverse V(L) gene segments in a South American opossum, Monodelphis domestica, is consistent with this marsupial being an exception to the pattern. To determine whether M. domestica is unique or the norm for marsupials, the V(H) and V(L) of an Australian possum, Trichosurus vulpecula, were characterized. The Ig repertoire in T. vulpecula is also derived from a restricted V(H) pool but a diverse V(L) pool. The V(L) gene segments of T. vulpecula are highly complex and contain lineages that predate the separation of marsupials and placental mammals. Thus, neither marsupial follows a pattern of coevolution of V(H) and V(L) gene segments observed in other mammals. Rather, marsupial V(H) and V(L) complexity appears to be evolving divergently, retaining diversity in V(L) perhaps to compensate for limited V(H) diversity. There is a high degree of similarity between the V(H) and V(L) in M. domestica and T. vulpecula, with the majority of V(L) families being shared between both species. All marsupial V(H) sequences isolated so far form a common clade of closely related sequences, and in contrast to the V(L) genes, the V(H) likely underwent a major loss of diversity early in marsupial evolution.

Conjunctival vaccination of the brushtail possum (Trichosurus vulpecula) with bacille Calmette-Guérin.

AIM: To determine the efficacy of conjunctival vaccination of captive brushtail possums (Trichosurus vulpecula) with bacille Calmette-Guérin (BCG), as measured by immunological responses to vaccination and response to intratracheal challenge with Mycobacterium bovis. METHODS: Nine adult male brushtail possums were vaccinated by the instillation of a suspension of BCG strain Pasteur 1173P2 into the conjunctival sac of each eye. Each drop contained approximately 2.5 x 105 colony forming units (cfu). At 8 weeks post-vaccination (pv) the vaccinated possums and 10 unvaccinated possums were challenged by intratracheal instillation of approximately 100 cfu of M. bovis. Cellular immune responses to bovine purified protein derivative (PPD) antigen were measured using the lymphocyte proliferation assay (LPA). Possums surviving to 50-51 days after challenge were euthanised and subjected to detailed post-mortem examination, including histopathology, to assess protection against tuberculosis. Sections of lung and spleen were cultured for M. bovis. RESULTS: No conjunctival inflammation or other adverse reactions to the administration of the vaccine were evident macroscopically. The vaccinated group showed a systemic cellular immune response to bovine PPD antigen at 4 and 8 weeks pv, and the response at 8 weeks was significantly greater than at 4 weeks (p<0.05). Conjunctival vaccination induced significant levels of protective immunity, measured as less mass of tuberculous lesions in lung (p<0.05) and less dissemination of disease in vaccinated compared with unvaccinated possums (p<0.05). CONCLUSIONS: Conjunctival vaccination with BCG induced a significant level of protective immunity against M. bovis infection in possums. This route of vaccination, together with intranasal aerosol vaccination, could be utilised in the delivery of an aerosolised vaccine using a device that sprays the vaccine suspension into the eyes and nose of possums.

Mapping of B cell epitopes on the zona pellucida 2 protein of a marsupial, the brushtail possum (Trichosurus vulpecula).

Immunocontraceptive vaccines against zona pellucida (ZP) proteins are being developed for brushtail possum (Trichosurus vulpecula) management in New Zealand. Mapping of B cell epitopes on the ZP2 protein of possums was undertaken in this study to define the antigenic regions that may be crucial to sperm-egg binding. The amino acid sequence of the full-length possum ZP2 protein (712 amino acids) was used to synthesize a complete set of 71 (15-mer) biotinylated peptides with an offset of five amino acids. The peptides were used in a modified enzyme-linked immunosorbent assay (ELISA) to identify continuous epitopes recognized by antibodies in the sera of possums immunized with recombinant possum ZP2 (rZP2) constructs. Seventeen continuous epitopes were located on possum ZP2 protein. Comparisons of the peptide binding pattern of antibodies in individual sera with the fertility status of the same immunized possums revealed three significant infertility-relevant peptide epitopes (amino acids 111-125, 301-315, and 431-445). One of these (amino acids 431-445) bound to possum spermatozoa from the caudal epididymis. The implications of these findings for developing immunocontraceptive vaccines for possum control are discussed.

Vaccination of brushtail possums, Trichosurus vulpecula, with Bacille Calmette-Guerin induces T lymphocytes that reduce Mycobacterium bovis replication in alveolar macrophages via a contact-dependent/nitric oxide-independent mechanism.

The permissiveness of alveolar macrophages from brushtail possums for the replication of Mycobacterium bovis was examined. Mycobacterium bovis replication was indirectly measured by assessing bacterial metabolism via the incorporation of [3-H]-uracil by bacilli released from lysed macrophages previously infected with mycobacteria. Alveolar macrophages allowed substantial replication of virulent M. bovis, in contrast to Bacille Calmette-Guerin (BCG) Pasteur, which replicated poorly. The addition of crude lymphokines enhanced the metabolic activity of phagocytosed M. bovis in possum macrophages. Possum lymphokines enhanced the ability of possum macrophages to generate reactive oxygen intermediates, measured by the reduction of nitroblue tetrazolium, which is indicative of an activation process. Similarly, the addition of recombinant possum TNF-alpha enhanced the permissiveness of alveolar macrophages for M. bovis. In contrast to mouse peritoneal macrophages, possum alveolar macrophages did not release significant levels of nitric oxide (NO) after stimulation with M. bovis and/or lymphokines. However, the uptake of virulent M. bovis by possum macrophages was associated with an enhanced ability of cells to release TNF-alpha, whereas very low levels of TNF-alpha were released after infection with BCG. The addition of a selective inhibitor of inducible NO synthase had no impact on the replication of M. bovis or BCG in possum macrophages in the presence or absence of lymphokines. Co-culturing infected possum alveolar macrophages with autologous blood mononuclear cells from BCG-vaccinated possums led to a significant decrease in the metabolic activity of intracellular M. bovis. This effect was contact dependent and NO independent and was mediated by a population of CD3+ cells. In addition, adding scavengers of reactive oxygen intermediates did not abrogate this phenomenon.