Molecular identification of interleukin-2 in the lymphoid tissues of the common brushtail possum, Trichosurus vulpecula.

The common brushtail possum (Trichosurus vulpecula) is an Australian marsupial. Here we describe the identification of possum interleukin-2 in mitogen-stimulated lymph node cells. We used a strategy of Rapid amplification of cDNA ends using probes designed from recently-sequenced marsupial genomes to identify the IL2 gene and then confirmed that IL-2 expression in possum immune tissue occurs in a similar manner to that in their eutherian counterparts. The predictive possum IL-2 peptide showed 28% and 35% amino acid sequence homology with the mouse and human IL-2 molecules, respectively, consistent with the divergence found within this cytokine family. Despite this low sequence identity, possum IL-2 still possessed the characteristic hallmarks of mammalian IL-2, such as a predicted signal peptide and conserved family motifs.

Uterine and vaginal insemination optimised in brushtail possums (Trichosurus vulpecula) superovulated with pregnant mare serum gonadotrophin and porcine luteinising hormone.

Artificial insemination of brushtail possums (Trichosurus vulpecula) is being developed as an assisted breeding model for endangered marsupials, as well as a bioassay for testing fertility control vaccines to manage overabundant populations. Procedures were optimised in animals superovulated with pregnant mare serum gonadotrophin (PMSG) and porcine luteinising hormone (pLH). Of three intervals examined, yields were maximal following uterine insemination at 27-29.5 h after pLH treatment (four eggs, two to three embryos per female). Compared with no insemination, uterine-inseminated animals ovulated 30-36 h rather than 28-34 h after pLH treatment. For the vaginal route, yields were maximal following insemination at 10-13 h after pLH treatment (six to seven eggs, four embryos per female) than at five other intervals, and when using acclimatised females during the autumn breeding season. This protocol was suitable for testing fertility control vaccines in April-June and was influenced by the housing location of animals, the presence of an active corpus luteum and PMSG batch, but not other factors (year of trial, Freund's adjuvant treatment, changes in bodyweight, dose of PMSG kg(-1)). Embryos developed to the eight- to 16-cell or unilaminar blastocyst stage after uterine or vaginal insemination, respectively. With the timing of artificial insemination optimised, new methods to synchronise or induce oestrus and ovulation are required to achieve year-round testing of fertility control vaccines or birth of offspring.

Humoral immunity to cholera toxin and ovalbumin in the brushtail possum (Trichosurus vulpecula).

AIM: To compare the humoral immune responses of female possums to ovalbumin (OVA) and cholera toxin (CT) administered using different routes and dosage regimes. METHODS: Three groups of mature female possums were immunised intragastrically (n=15) or surgically (intraileal, n=4; Peyer's patches, n=4). Levels of antibodies to OVA and CT in serum, ileal, vaginal, oviduct and uterine secretions were measured by indirect enzyme-linked immunosorbent assay (ELISA). All manipulations were conducted under general anaesthesia. RESULTS: Antibodies to CT increased significantly in serum (p<0.001) and in lateral vaginal secretions (p<0.05) after intragastric immunisation using 250-1000 microg CT. Simultaneous intragastric administration of CT with 3 mg OVA did not significantly increase antibody levels to OVA. When OVA (3 mg) and CT (250 microg) were injected into the ileum, followed by intragastric administration 4 weeks later, antibody levels to CT were significantly (p<0.05) increased in serum, uterine and medial vaginal secretions, and antibody levels to OVA were significantly (p<0.05) increased in serum and medial vaginal secretions. When OVA (3 mg) with Incomplete Freund's adjuvant was injected directly into Peyer's patches, followed by peritoneal administration 4 weeks later, antibody levels in serum, ileal and reproductive-tract secretions were generally higher than those that resulted from the other immunisation treatments. CONCLUSIONS: Administration of OVA induced an antibody response in serum and in reproductive and alimentary tract secretions when administered with CT directly into the ileum, or when injected into Peyer's patches and peritoneum. CT was a potent enteric immunogen.

Fertility of brushtail possums (Trichosurus vulpecula) immunised against sperm.

The brushtail possum (Trichosurus vulpecula) is a major pest species in New Zealand. Research to develop a possum-specific method of immunologically-based fertility control (immunocontraception) has begun. This study examined the effect of immunization against sperm on the fertility of possums. Possums were assigned to 16 mating groups (two females and one male per group) before the start of the breeding season. One female in each group and eight males were injected subcutaneously with 5 x 10(7) sperm in complete Freund's adjuvant. Animals were given booster injections containing 5 x 10(7) sperm in incomplete Freund's adjuvant 4 and 12 weeks later. Control animals received adjuvants emulsified with phosphate buffered saline. Serum and sperm samples were collected monthly and the number of offspring recorded. Reproductive tract samples were collected from eight immunized and eight control possums of each sex at the end of the breeding season. Samples were screened for sperm antibody binding by an indirect possum sperm ELISA. Increased sperm antibody binding was detected in sera of 22 (15 females and seven males) of the 24 animals immunized against sperm. Immunization significantly reduced the proportion of females producing offspring (2/16 immunized versus 12/16 control) but had no effect on the fertility of males. Immunization increased sperm antibody binding activity in vaginal secretions of female and in prostatic tissue of male possums. Results indicate that there are antigens in possum sperm with immunocontraceptive potential for possum control.