B lymphocyte activities in the opossum, Trichosurus vulpecula : a thesis presented in partial fulfilment of the requirement for the degree of Doctor of Philosophy at Massey University

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Massey University
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The evolution of vertebrate immunity from the level of the protochordates to that of the metatherians is reviewed. Using standard methods IgG, IgM and IgA were isolated from the serum or intestinal fluid of the Australian brush-tailed opossum, Trichosurus vulpecuia. These were characterized in terms of their molecular weights, amino acid and carbohydrate compositions and values for their concentrations in serum were calculated. Two forms of IgG were seen which differed in their abilities to bind to insoluble matrices and also in their molecular weights. No antigenic differences were seen between them on analysis by agar diffusion . The molecular weight of the IgA seen in intestinal fluid and results from its analysis by agar diffusion suggest that the molecule may lack secretory component. B lymphocytes were identified by their surface immunoglobulin and their complement and Fc receptors. The number of these cells in blood and various lymphoid tissues of T.vulpecula was found to be similar to the values reported for mice and humans. Lymphocyte fractionation on nylon wool columns confirmed that the markers employed were associated with an adherent cell population. Blood lymphocytes were stimulated in vitro with a range of mitogens and the degree of transformation achieved with each was assessed by the cells uptake of tritiated thymidine. Insoluble concanavalin A, pokeweed mitogen and lipopolysaccharide, in that order, were the most effective of the mitogens used on unfractionated blood lymphocytes. These three mitogens were further used in studies in which nylon wool fractionation of blood lymphocytes was used to prepare B cell- and T cell-enriched cultures. Lipopolysaccharide was the only mitogen to stimulate B cells more than T cells. Insoluble concanavalin A consistently stimulated T cells to a greater extent than B cells as did pokeweed mitogen. The ultrastructure of mitogen-stimulated cells was studied by electron microscopy and it was shown that lipopolysaccharide induced the formation of plasmablasts which resembled those of eutherians. Mitogen-stimulated cells were also analysed for their production of immunoglobulins, the levels of de novo synthesised materials being measured by their incorporation of isotope-labelled leucine provided in the culture medium. Both secreted and intracellular proteins were measured in this way. Lipopolysaccharide, pokeweed mitogen and insoluble concanavalin A all induced significantly increased levels of 19S and 7S secreted proteins, these proteins being separated by gel filtration. Pokeweed mitogen induced the synthesis of significantly increased levels of both 19S and 7S intracellular proteins, while lipopolysaccharide and insoluble concanavalin A significantly increased the levels of 19S protein only. The presence of IgM and IgG in the 19S and 7S fractions was shown by their precipitation with class-specific antisera. The immune responses of T.vulpecula to a particulate and a soluble antigen were compared with those of rabbits to the same antigens. Sheep erythrocytes, at two dose levels, were injected intravenously. The responses of opossums to 5x109 erythrocytes were appreciably more rapid than those of the rabbits. The responses of the two species to 25x109 erythrocytes were similar in the titres attained and the time taken to do so. The distribution of haemagglutinating activity between IgM and IgG was studied and found to be essentially the same for both species for both levels of antigen. The responses of opossums to bovine serum albumin injected intramuscularly with Freund's adjuvants were similar to those of rabbits. It is concluded that the B cell-dependent immune functions of T.vulpecula are as efficient as those of other metatherians and compare favourably with those of eutherians.
Possum, B lymphocytes, B cells, Trichosurus vulpecula, Brushtail possum, Vertebrate immunity, Antigens, Beta lymphocytes