Marsupial Immunology

Baseline haematology, blood chemistry and acute phase protein parameters have not previously been published for free-ranging eastern grey kangaroos (Macropus giganteus). Eight eastern grey kangaroos, including three adult males, three adult females and two subadult males from two different populations, were examined. Assays assessed the antibacterial activity of kangaroo serum against one Gram-positive and three Gram-negative bacteria. The kangaroo serum had a strong antibacterial response to Klebsiella pneumoniae, and moderate responses to Escherichia coli and Staphylococcus aureus. The presence and level of acute phase proteins, haptoglobin and serum amyloid A in kangaroos was investigated. Haptoglobin and serum amyloid A were present in kangaroo serum, but only haptoglobin was elevated in a kangaroo with capture myopathy and necrotic wounds. The findings of this study provide preliminary data on health parameters of free-ranging eastern grey kangaroos. These parameters can be used to assist in assessing health in free-ranging populations.

Marsupials are born immunologically premature, relying on cells and molecules in maternal milk for immune protection. Both immunoglobulin and complement proteins have been identified in marsupial milk, but the expression of specific complement proteins remains largely unexplored. We report partial cDNA sequences for two complement-activating proteins, C3, C1r, CFP and MASP2, in liver tissues from red-tailed phascogale (Phascogale calura). Conservation of functionally relevant motifs were identified in the translated cDNA sequences from phascogale C3, CFP and MASP2 and their eutherian homologues. Gene expression of representative molecules from each of the major complement pathways was also investigated in whole body tissues from 1 to 18 day old animals and liver tissues from 31-day to 14-month old animals. Average complement expression in whole bodies and liver tissues of C1r, CFP, MASP2 and C3 increased significantly in juveniles compared to pouch young, presumably due to the maturation of the young's own complement system. Comparing expression in liver tissues only, we found that the average CFP expression were higher in pouch young compared to juveniles, while results were still statistically similar to the average expression of all tissues for C1r, MASP2 and C3. The average complement expression then significantly decreased as the animals aged into adulthood.

Background. Reference genes serve an important role as an endogenous control/s-tandard for data normalisation in gene expression studies. Although reference genes have recently been suggested for marsupials, independent analysis of reference genes on different immune tissues is yet to be tested. Therefore, an assessment of reference genes is needed for the selection of stable, expressed genes across different marsupial tissues. Methods. The study was conducted on red-tailed phascogales (Phascogale calura) using five juvenile and five adult males. The stability of five reference genes (glyceraldehyde-3-phosphate dehydrogenase, GAPDH ; β-actin, ACTB; 18S rRNA, 18S; 28S rRNA, 28S; and ribosomal protein L13A, RPL13A) was investigated using SYBR Green and analysed with the geNorm application available in qBase PLUS software. Results. Gene stability for juvenile and adult tissue samples combined show that GAPDH was most stable in liver and lung tissue, and 18S in small intestine and spleen. While all reference genes were suitable for small intestine and spleen tissues, all reference genes except 28S were stable for lung and only 18S and 28S were stable for liver tissue. Separating the two age groups, we found that two different reference genes were considered stable in juveniles (ACTB and GAPDH) and adults (18S and 28S), and RPL13A was not stable for juvenile small intestine tissue. Except for 28S, all reference genes were stable in juvenile and adult lungs, and all five reference genes were stable in spleen tissue. Discussion. Based on expression stability, ACTB and GAPDH are suitable for all tissues when studying the expression of marsupials in two age groups, except for adult liver tissues. The expression stability between juvenile and adult liver tissue was most unstable, as the stable reference genes for juveniles and adults were different. Juvenile and adult lung, small intestine and spleen share similar stable reference genes, except for small intestine tissues where all reference genes were stable in adults but RPL13A was not suitable in juveniles.

Background: The red-tailed phascogale is a 'Near Threatened' dasyurid marsupial. Males are semelparous and die off shortly after the breeding season in the wild due to a stress-related syndrome, which has many physiological and immunological repercussions. In captivity, males survive for more than 2 years but become infertile after their first breeding season. Meanwhile, females can breed for many years. This suggests that captive males develop similar endocrine changes as their wild counterparts and undergo accelerated aging. However, this remains to be confirmed. The health status and immune function of this species in captivity have also yet to be characterized. Results: Through an integrative approach combining post-mortem examinations, blood biochemical and hematological analyses, we investigated the physiological and health status of captive phascogales before, during, and after the breeding season. Adult males showed only mild lesions compatible with an endocrine disorder. Both sexes globally maintained a good body condition throughout their lives, most likely due to a high quality diet. However, biochemistry changes potentially compatible with an early onset of renal or hepatic insufficiency were detected in older individuals. Masses and possible hypocalcemia were observed anecdotally in old females. With this increased knowledge of the physiological status of captive phascogales, interpretation of their immune profile at different age stages was then attempted. During the breeding season, males developed a stress leukogram characterized by a marked lymphopenia, further aggravated by a severe leukopenia after the breeding season. To determine whether these changes were limited to the peripheral blood or had more profound implications, histopathology of the spleen was performed opportunistically. Adult males showed white pulp atrophy, at various degrees. The atrophy was mainly lymphoid and more severe in 1.5-year-old males than in 3.5-year-old females. These results suggest that captive males undergo accelerated immunosenescence.

Red-tailed phascogale (Phascogale calura) pouch young at birth were relatively underdeveloped in comparison with their eutherian counterparts, and the lymphoid tissues of the immune system were found to be histologi-cally immature. The phascogale thymus rapidly developed in the first few days of pouch life and was quickly populated with lymphocytes. By the end of pouch life, involution of the thymus was underway. The bone marrow started to develop in the early stage of pouch life, although adipocytes and megakaryocytes were not observed until slightly later. The liver was hematopoietic from birth and reached histological maturity toward the end of pouch life. The lymph nodes were difficult to detect macroscopically because of their small size, but were easily identified microscopically later in pouch life, particularly in the mesentery, and these lymph nodes exhibited germinal centers by the end of pouch life. The early spleen was pre-dominately mesenchymal, but exhibited some erythropoiesis. Follicles with well-developed germinal centers were not observed until the latest stage of pouch life. Although intraepithelial lymphocytes were detected in the intestines early in pouch life, the discrete lymphoid aggregates and Peyer's patches characteristic of the gut-associated lymphoid tissue (GALT) were not detected until later in pouch life. This is the first report of histological development in phascogale pouch young, as well as the first report of the thymus, bone marrow, and lymph nodes in this dasyurid species at any age. Anat Rec, 299:207–219, 2016. V C 2015 Wiley Periodicals, Inc.