Lauren J. Young

Immunobiology of mycobacterial infections in marsupials

Mycobacterial infections of marsupials are important for two reasons. Firstly, the Australian brushtail possum (Trichosurus vulpecula) serves as the major wildlife reservoir for Mycobacterium bovis in New Zealand and secondly, M. avium is a significant cause of disease in endangered marsupial species held in captivity. Marsupials are highly susceptible to specific mycobacterial infections which may be linked to deficiencies in their cellular immunity. Histopathological inspection of affected tissues indicates that, unlike most eutherians, marsupials are unable to wall off infection sites, resulting in formation of satellite lesions and generalised disease. This review examines possible reasons for the high susceptibility of marsupials to mycobacterial infections and investigates the prospects for developing vaccines to control these diseases.

Culture and Stimulation of Tammar Wallaby Lymphocytes

We describe the culture and stimulation of lymphocytes from the model marsupial, the tammar wallaby (Macropus eugenii). We also describe the capacity of tammar wallaby lymphocytes isolated from blood, spleen and lymph nodes to produce soluble immunomodulatory factors. Culture conditions were optimized for mitogen-driven stimulation using the plant lectin phytohaemagglutinin (PHA). Products secreted by stimulated cells were harvested and crudely fractionated before they were added back to freshly isolated lymphocytes. Using the 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) assay, both stimulatory and inhibitory bioactive factors were detected in serum-free supernatants harvested from mitogen-treated peripheral blood mononuclear cells. This paper describes the capacity of leukocytes of the tammar wallaby to respond to mitogenic stimulation and to produce soluble, low-molecular-weight bioactive molecules that possess cytokine-like activity.

The development of the immune tissues in marsupial pouch young

Current knowledge of the development of the marsupial immune system, particularly in the context of lymphoid tissue development and the appearance of lymphocytes, has been examined and limitations identified. While primary lymphoid tissues like the thymus have been extensively studied, secondary lymphoid tissues such as the spleen and lymph nodes have been examined to a lesser extent, partly due to the difficulty of macroscopically identifying these structures, particularly in very small neonates. In addition, little research has been conducted on the mucosal‐associated lymphoid tissues; tissues that directly trap antigens and play an important role in the maturity of adaptive immune responses. Research on the development of the marsupial immune tissues to date serves as a solid foundation for further research, particularly on the mechanisms behind the development of the immune system of marsupials. With the recent sequencing and annotation of whole marsupial genomes, the current wealth of sequence data will be essential in the development of marsupial specific reagents, including antibodies, that are required to widen our specific knowledge of the complex marsupial immune system and its development. J. Morphol. 275:822–839, 2014.

Marsupial immunology bounding ahead

Abstract. Marsupial immune responses were previously touted as ‘primitive’ but we now know that the marsupial immune system is complex and on par with that of eutherian mammals. In this manuscript we review the field of marsupial immunology, focusing on basic anatomy, developmental immunology, immunogenetics and evolution. We concentrate on advances to our understanding of marsupial immune gene architecture, made possible by the recent sequencing of the opossum, tammar wallaby and Tasmanian devil genomes. Characterisation of immune gene sequences now paves the way for the development of immunological assays that will allow us to more accurately study health and disease in marsupials.

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.

Molecular characterisation and expression of interleukin-6 and interleukin-6Δ2 in the Tammar wallaby (Macropus eugenii).

The pro-inflammatory cytokine, Interleukin-6 (IL-6), has not yet been fully characterised in the model macropod, Macropus eugenii, due to incomplete sequence information in publically available genome databases. Using a Rapid Amplification of cDNA Ends strategy we have confirmed the expression and complete nucleotide sequence for this molecule in lymph node tissue and activated leukocytes. Structural conservation of the mature wallaby IL-6 molecule was high when compared with human IL-6, although there was only 34% amino acid sequence identity with the human IL-6 peptide, consistent with reports of the evolutionary divergence of this cytokine. We also report the discovery of MeIL-6Δ2, a splice variant missing exon 2, which directly translates to a truncated non-functional peptide, but which may also code for an alternative peptide that is translated downstream of the canonical IL-6 start site. This putative gene product is predicted to maintain some, if not all, of the functions of macropod IL-6 and is the first IL-6 isoform reported outside of eutherian mammals.

A longitudinal study of changes in blood leukocyte numbers in the tammar wallaby, Macropus eugenii

Changes in leukocyte numbers were monitored over a 3-year period in a small group of captive tammar wallabies, Macropus eugenii, maintained in the animal research facilities at Macquarie University (NSW, Australia). The neutrophil to lymphocyte ratio (N/L), a commonly used parameter in the assessment of health status in wildlife populations, was not useful when applied between animal populations but did reliably predict changes within individual animals and between animals within the study cohort. This study also demonstrated the importance of obtaining haematological values from animals on more than one occasion to ensure that differential cell counts from asymptomatic individuals do not unduly influence the determination of reference values.

Detection of an active complement system in red-tailed phascogales (Phascogale calura)

Very few assays that are used to assess the status of mammalian immunity have proved useful for assessment of marsupial health and/or diagnosis of disease. This is largely due to the lack of species cross-reactive reagents that underpin such experiments. To begin to address this deficit, we describe the activation of classical and alternative complement pathways of red-tailed phascogales (RTP; Phascogale calura). Using standard haemolytic assays, the existence of both complement pathways were established in RTP serum based on its ability to lyse unsensitised rabbit erythrocytes (RbE) and sensitised sheep erythrocytes (SE), respectively. The alternative complement pathway assays were conducted using pooled serum of male and female RTPs, and the remaining RTP sera were opportunistically used to test the presence of a functional classical complement system in individual animals, a first in non-eutherian animals. Observations from this study suggest that the activation of these two complement pathways in RTPs are comparable to that seen in other mammals. Since this assay was able to be used on very small samples of blood, it could serve as a useful tool to gather data for comparative immunological studies and to further our knowledge of the mechanisms of immunity available to marsupial young.

Immunocytochemical identification of CD5 positive peripheral blood lymphocytes in four marsupial species

Abstract Immunocytochemical analysis of peripheral blood mononuclear cells was undertaken using a streptavidin biotin–horseradish peroxidase method to detect CD5 positive lymphocytes from the blood of several marsupial species. A monoclonal antibody raised to a conserved peptide sequence of the human CD5 antigen positively labelled lymphocytes in freshly isolated peripheral blood mononuclear cells of the tammar wallaby (Macropus eugenii), the long-footed potoroo (Potorous longipes), the long-nosed potoroo (Potorous tridactylus) and the rufous hare-wallaby (Lagorchestes hirsutus). A polyclonal anti-CD3 antibody also positively labelled circulating lymphocytes from the tammar wallaby. Whereas previous studies using flow cytometry reported labelling of T cells in koala lymphocyte preparations using a polyclonal anti-CD3 antibody, there have been no other reports of marsupial blood immunophenotyping. The current study extends the known applications of monoclonal anti-CD5 and polyclonal anti-CD3 antibodies to blood lymphocytes of small wallaby species using an immunocytochemical slide technique that is simple, can be processed within a day and requires no dedicated large equipment.

Sequences and expression of pathway-specific complement components in developing red-tailed phascogale (Phascogale calura).

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 youngs 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.