Lymphocyte proliferation and antibody responses to select antigens in the brushtail possum

The aim of this study was to compare the in vitro lymphocyte proliferation and antibody responses to ovalbumin (OVA) and keyhole limpet haemocyanin (KLH) after being given intragastrically or subcutaneously to female possums. Fifty four mature female possums were assigned to nine treatment groups (6 possums /group). For intragastric treatments (6 groups), possums received either 2 mg KLH, 2 mg KLH + 100 µg cholera toxin (CT), 2 mg OVA, 2 mg OVA + 100 µg CT, 100 µg CT or phosphate buffer saline (PBS). For subcutaneous administration (3 groups), possums received either 2 mg KLH incorporated in Freund's adjuvant (FA), 2 mg OVA with FA or FA alone, where complete Freund's adjuvant (CFA) was used at week 0, and incomplete Freund's adjuvant (IFA) was used at weeks 2 and 4. Treatments were administered and blood samples collected at 2 weekly intervals and all animals were euthanised at week 6 of the trial to allow for collection of samples for the lymphocyte blastogenic test (LBT) and ELISA. Intragastric administration of KLH or OVA did not enhance antigen-specific lymphocyte blastogenic activity from blood, mesenteric or axillary lymph nodes. The addition of CT as a mucosal adjuvant enhanced lymphocyte proliferative responses to KLH (and to a lesser extent OVA) from mesenteric and axillary lymph nodes (p<0.01 and p<0.05 respectively). Subcutaneous administration of KLH or OVA with FA caused significantly higher antigen specific proliferation of blood lymphocytes over repeated time points at week 4 (p<0.001) and week 6 (p<0.05 and p<0.001 respectively) as compared to preimmunisation levels or control (FA alone). Similarly, sensitised lymphocytes from mesenteric and axillary lymph nodes gave antigen-specific blastogenic responses to KLH and OVA in vitro which were higher after subcutaneous administration with FA than either FA alone (p<0.05) or the intragastric treatments (p< 0.05). When KLH or OVA were given alone intragastrically they failed to induce antibody responses in the plasma, even though KLH was able to induce antigen-specific antibodies in duodenal and certain reproductive tract secretions. Cholera toxin was a good immunogen as it stimulated the production of anti-CT antibodies in plasma at weeks 4 (p<0.05) and 6 (p<0.01) and in mucosal secretions (p<0.05) compared to preimmunisation levels and control. However, as an adjuvant given intragastrically CT did not enhance the antibody response to KLH or OVA in plasma or mucosal secretions. Subcutaneous administration of the antigens KLH or OVA with FA induced high levels of plasma antibodies at weeks 2, 4 and 6 (p<0.001) as compared to both preimmunisation levels and other treatment groups, and in all examined mucosal secretions at slaughter (p<0.001). In the treatment groups given KLH intragastrically and OVA plus CT intragastrically, levels of antibodies detected in plasma and mucosal secretions were found to be positively correlated with lymphocyte blastogenic activity. It appears that soluble or non replicative proteins are not strongly immunogenic when given intragastrically but can cause immune responses in distant mucosal sites. The responses are less than those following a subcutaneous injection of antigens with Freund's adjuvant. The results suggest that more complex antigens are capable of inducing better immune responses than soluble easily-degradable antigens. The association between lymphocyte blastogenic activity and levels of antibodies in some treatment groups suggests that the antibody response is T cell dependent.