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Expressed coding sequences for interleukin-6 (IL-6) and interleukin-6 receptor α (IL-6R) were examined in five marsupial species. Full length expressed coding sequences for IL-6 and IL-6R were identified and characterized in the gray short-tailed opossum (Monodelphis domestica). For IL-6, ∼225 bp fragments of the mRNA sequence were identified in the red-tailed phascogale (Phascogale calura), kultarr (Antechinomys laniger), and stripe-faced dunnart (Sminthopsis macroura), while ∼563 bp fragments of mRNA encoding IL-6R were identified in the red-tailed phascogale, kultarr, stripe-face dunnart and fat-tailed dunnart (Sminthopsis crassicaudata). Relative expression levels of IL-6 and IL-6R were examined in the heart, muscle, lung, liver, spleen and kidney of adult red-tailed phascogales, and IL-6 gene expression was found to be significantly higher in the lung and spleen than the other tissues examined, while the expression of IL-6R was significantly higher in the liver, lung and spleen. These results now serve as a reference point for examining the role and levels of IL-6 and IL-6R in the health and disease of these marsupial species. The pro-tumorigenic nature of IL-6 is of particular interest, and the identification of these IL-6 and IL-6R coding sequences provides a platform for further work to evaluate the potential role of IL-6 in marsupial cancers.

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.

In mammals, interleukin-21 is a member of the common gamma chain cytokine family that also includes IL-2, IL-4, IL-7, IL-9 and IL-15. IL-21 has pleiotropic effects on both myeloid and lymphoid immune cells and as a consequence, the biological actions of IL-21 are broad: regulating both innate and adaptive immune responses and playing a pivotal role in antiviral, inflammatory and antitumour cellular responses. While IL-21 genes have been characterized in mammals, birds, fish and amphibians, there are no reports for any marsupial species to date. We characterized the expressed IL-21 gene from immune tissues of two macropod species, the tammar wallaby (Macropus eugenii), a model macropod, and the closely related endangered bridled nailtail wallaby (Onychogalea fraenata). The open reading frame of macropod IL-21 is 462 nucleotides in length and encodes a 153-mer putative protein that has 46% identity with human IL-21. Despite the somewhat low amino acid conservation with other mammals, structural elements and residues essential for IL-21 conformation and receptor association were conserved in the macropod IL-21 predicted peptides. The detection of IL-21 gene expression in T-cell-enriched tissues, combined with analysis of the promotor region of the tammar wallaby gene, suggests that macropod IL-21 is expressed in stimulated T cells but is not readily detected in other cells and tissues. The similarity of gene expression profile and functionally important amino acid residues to eutherian IL-21 makes it unlikely that the differences in B- and T-cell responses that are reported for some marsupial species are due to a lack of important functional residues or IL-21 gene expression in this group of mammals.

Studies of gene diversity are used to investigate population dynamics, including immunological fitness. Aside from the selection of an appropriate gene target, an important factor that underpins these studies is the ability to recover viable DNA samples from native animals that are protected, threatened or difficult to sample or locate such as the bare-nosed wombat (Vombatus ursinus). In this study, we used genomic DNA extracted from muscle tissue samples and also identified the optimal method to extract DNA from fresh wombat scat samples to enable further analyses to be performed using non-invasive techniques. The DNA was probed via the polymerase chain reaction using previously targeted marsupial Major Histocompatibility Complex (MHC) gene primers. These genes are highly variable and associated with binding and presentation of pathogens in the immune system. Twenty-three unique MHC Class II DAB V. ursinus gene sequences were translated to 21 unique predicted peptide sequences from 34 individual tissue or scat samples. Vombatus ursinus MHC Class II DAB gene and peptide sequences were most similar to other marsupial DNA and peptide sequences. Further analysis also indicated the likelihood of MHC Class II DAB family membership through motif identification. Additional sampling is required to assess the full level of diversity of MHC Class II DAB genes among V. ursinus populations; however, this study is the first to identify MHC genes in a wombat and will advance immunological and disease studies of the species.

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. Conclusions Functional studies are now needed to characterize the underlying mechanisms leading to immunosenescence in marsupials. Semelparous dasyurids present great potential for studying the effects of sex and stress on immunity in marsupials. Characterization of these immune-endocrine interactions may help refine veterinary treatment plans, husbandry protocols and conservation programs to maintain the health of captive and wild populations.

Cells expressing the surface markers CD3, CD4, CD79b, IgM, MHC class II, and ModoUG (nonclassical MHC class I) were detected in red-tailed phascogale tissues using immunohistochemistry, and the appearance and localization of cells observed here was consistent with previous observations in other marsupial species. CD3+ cells were first detected at one day postpartum (dpp) in the thymus, followed by ModoUG+ cells at 5-7 dpp in the thymus and lymph nodes. CD79b+ cells were first detected at 12-14 dpp in bone marrow, spleen, and lymph nodes. IgM+ cells were first detected at 12-14 dpp in thymus, bone marrow, spleen, and lymph nodes. MHC class II+ cells were first detected at 12-14 dpp in thymus, bone marrow, and lymph nodes. CD4+ cells were detected in adult thymus and spleen only. The presence of the mature immune cell populations and their localization to characteristic T and B cell zones in mature lymphoid tissues with normal histological structure indicates that red-tailed phascogales develop immunocompetence by the end of pouch life. Anat Rec, 302:1985-2002, 2019. © 2019 American Association for Anatomy.

Objective Marsupials suffer from an increasing number of stressors in this changing world. Functional studies are thus needed to broaden our understanding of the marsupial immune system. The red-tailed phascogale (Phascogale calura) is a small Australian marsupial previously used in descriptive immunological studies. Here, we aimed to develop functional assays by isolating and stimulating blood and spleen mononuclear cells in vitro. Results While peripheral blood mononuclear cell (PBMC) were relatively easy to isolate, only 105 mononuclear cells (> 90% purity and > 75% viability) could be recovered from the spleen, independently of the sex and age of the animal or the centrifugation time and speed tested. The pores of the mesh sieve used for tissue homogenization might have been too big to yield a single cell suspension. Nevertheless, in spite of the overall low number of cells recovered, PBMC and splenic mononuclear cells were successfully activated in preliminary trials with phytohemaglutinin. This activation state was evidenced by a change in shape and the presence of small cell aggregations in the mitogen-stimulated cultures. A non-radioactive colorimetric assay was also performed to confirm cell proliferation in these wells. This work highlights the importance of developing and reporting detailed methodological protocols in non-traditional research species.

Expressed coding sequences were identified for the T cell receptors TCRα, TCRβ, TCRγ, TCRδ, TCRμ, and co-receptors CD3e, CD4, and CD8α in the red-tailed phascogale (Phascogale calura). Expression of these genes was examined in the developing thymus and spleen of pouch young and juvenile individuals to detect any changes in expressionrelative to developmental stages. Transcripts of CD3e, CD4, TCRδ and TCRμ were first detected in the thorax at 1 day postpartum (dpp), and transcripts of all target genes were detected in the thymus from 3 dpp, and the spleen from 17 dpp. The relative expression of all target genes changed significantly over the course of pouch life and was associated with histological milestones of both the thymus and the spleen. Peak gene expression was observed early in pouch life in the thymus, with a decrease in expression associated with timing of thymic involution, while expression peaked later in the spleen and was associated with the initial differentiation of the red and white pulp and a population of T cells forming the periarterial lymphatic sheath (PALS). Active expression of these target genes within the thymus and spleen indicates that the young marsupials are actively developing immunocompetence during pouch life.

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.

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. © 2014 Wiley Periodicals, Inc.

Interleukin-2 (IL-2) is an important regulator of cellular immunity in mammals. For many years, our inability to identify the expression of this cytokine in marsupials hindered our capacity to progress studies in metatherian immunology. Here, we report the use of molecular techniques to characterise the IL-2 gene for the tammar wallaby (Notamacropus eugenii) and the Tasmanian devil (Sarcophilus harrisii), which allowed the prediction of the structure and probable functions of the IL-2 proteins of these species. Deduced marsupial IL-2 proteins show considerable sequence identity to each other and to common brushtail possum (Trichosurus vulpecula) IL-2 (≥65%) but shared only 35% (tammar wallaby) and 32% (Tasmanian devil) identity with human IL-2. This difference means that reagents used to study IL-2 in human and other eutherians are unlikely to cross-react with marsupials. As a key step in furthering our ability to study cellular immune responses in marsupials and, more specifically, the susceptibility of macropodoid marsupials to intracellular pathogens, a polyclonal antibody was designed for the detection and future investigation of tammar wallaby IL-2 protein expression. The molecular data and polyclonal antibody described herein will support our development of gene probes and immunological reagents that will aid studies of infection and disease in marsupials.

Background: The Eastern and Spotted-Tailed Quolls are “near threatened” Australian dasyurid marsupials that have undergone significant reduction in their geographic ranges in the past 200 years. Captive breeding and research colonies now exist, allowing further efforts to more fully understand the physiology of these carnivorous species. Objectives: The aims of the study were to provide a more detailed study of blood chemistry and differential WBC counts for Eastern and SpottedTailed Quolls, and to determine the influence by 3 biologic factors. Methods: Blood samples were taken from conscious, captive, healthy Eastern Quolls. A small number of samples from Spotted-Tailed Quolls were also available and were included in the study for comparison. Blood chemistry and differential WBC counts were compared to determine season-, age-, and sex-related differences. Results: For many of the analytes, blood chemistry results were comparable to other marsupial ranges, and no significant differences between sexes were detected ( P> .05). Seasonal differences were determined for total bilirubin, glucose, creatinine, and potassium concentrations in the Eastern Quoll. Generally, higher concentrations of these analytes were observed in the summer; however, amylase activity was significantly higher in autumn (southern hemisphere). Eastern Quolls one year of age and younger had significantly (P < .05) higher ALP activities than older animals. Conclusions: The normal ranges determined in this study can be used to assess clinical health of quolls and will assist with captive management and future reintroduction programs to the wild.

Lyn is a tyrosine kinase molecule required for modulation of signalling cascades in cell populations including B lymphocytes of the mammalian immune system. We have characterised the coding domain of the marsupial lyn gene of two macropod marsupials; the Tammar Wallaby (Macropus eugenii) and the Bridled Nailtail Wallaby (Onychogalea fraenata) and show the co-expression of two Lyn isoforms in cells and tissues of these and three other marsupials (Brush-tail Possum, Trichosurus vulpecula; American Grey Short-tailed Opossum, Monodelphis domestica and Red-tailed Phascogale, Phascogale calura). The predicted Lyn proteins (LynA and LynB) were highly conserved across vertebrate species, with amino acid identities of 94% with their human orthologues and conservation of key tyrosine kinase motifs that suggests that marsupial Lyn most likely functions in cell signalling. Comparison of our cDNA data to annotations for Lyn transcripts (available through the Ensembl Genome Browser) for the Tammar Wallaby confirm splice sites for a number of exons in the wallaby transcript that are missing from the current annotation. This is the first report of the expression of kinase signalling molecules that influence immunity in metatherian mammals and provides key information to support ongoing studies of immune regulation in marsupials.

Antimicrobial substances in serum include circulating complement proteins and acute phase proteins (APPs). We identified gene sequences for APPs, haptoglobin (Hp), C-reactive protein (CRP) and serum amyloid A (SAA) in marsupial genomes. Hp and SAA levels were measured in red-tailed phascogale ( Phascogale calura ) sera using commercially available assays. Hp levels were higher in males than females, while SAA levels suggest the phascogales used in this study were healthy. Serum was co-cultured with four bacterial species. Bacterial growth was inhibited after incubation at 37 °C, however effectiveness differed with bacteria and incubation time. The least amount of bacterial growth was noticed after introduction to K. pneumoniae , and most when introduced to P. aeruginosa . Despite marsupials not having mature immune tissues at birth, and unable to mount specific immune responses, this study suggests other immune strategies, such as APPs in serum likely aid marsupials in their defence against pathogens.

Interleukin-10 is an immunomodulatory cytokine that has been implicated, along with IFN-γ, in the disease sequelae of mycobacterial infection. In order to investigate the role of IL-10 in marsupial disease models we sequenced and characterised the IL10 gene in the tammar wallaby (Macropus eugenii) and rufous hare-wallaby (Lagorchestes hirsutus). An isoform IL-10Δ3, in which an in-frame deletion of exon 3 occurs, was discovered in both macropod species. Analysis of wallaby and other reported marsupial IL-10 homologs suggests that while marsupial IL-10 is comparable to that of human IL-10, the predicted IL-10Δ3 protein may play a more complicated role in the modulation of IL-10-directed responses. Expression of the canonical gene and splicing variant was confirmed in both wallabies, and the rufous hare-wallaby showed differential expression across lymph node, spleen and liver, with isoform expression detected in the lymph node. This characterisation and expression of IL-10 in de novo tissues provides a basis for further study into the role of IL-10 in disease models in marsupials.

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.

In this study, T and B lymphocytes were identified in the lungs of two potoroid marsupial species, the endangered Long-footed Potoroo (Potorous longipes) and the Long-nosed Potoroo (Potorous tridactylus). Using species cross-reactive antibodies, plasma cells, B cells and T cells were located in the alveolar septae and lymphocyte clusters were found adjacent to blood vessels and bronchioles. Bronchus-associated lymphoid tissue (BALT), resembling that of other mammals, was present within the bronchiolovascular bundles. Macrophages were occasionally visible in the alveoli, and areas of necrotic tissue in the Long-footed Potoroo contained collections of phagocytic cells, presumably macrophages, which contained acid-fast material and unidentifiable debris. This is the first report of BALT in Potoroo species and an important application of the only available antibodies that can reliably identify lymphocytes in marsupial species.

The granules of circulating leukocytes contain reactive oxygen species that are important components of host defence against bacterial invasion. We report the capacity of marsupials and monotremes to mount such a defence in a manner similar to their eutherian relatives. Using the nitroblue tetrazolium (NBT) test, reactive oxygen species were detected in the peripheral blood cells of five captive marsupial species (the tammar wallaby, Macropus eugenii, the Rufous hare wallaby, Lagorchestes hirsutus, the Brush-tailed bettong, Bettongia penicillata, the Long-footed potoroo, Potorous longipes, and the Long-nosed potoroo, Potorous tridactylus). The study included animals that were clinically healthy and those that were affected by mycobacterial disease. Animals in poor health elicited weak responses, consistent with the NBT test being used as a diagnostic assay for immunodeficiency. The NBT slide assay was also applied to platypus (Ornithorhyncus anatinus) and short-beaked echidna (Tachyglossus aculeatus) granulocytes to confirm the applicability of the test to this unique group of mammals.

Schistosomiasis is a chronic and debilitating disease caused by blood flukes (digenetic trematodes) of the genus Schistosoma. Schistosomiasis japonica, a zoonosis caused by Schistosoma japonicum, is endemic to the Philippines and China. We utilised a 22,575 feature custom oligonucleotide DNA microarray designed from public domain databases of schistosome-expressed sequence tags to explore differential gene expression between the Philippine (SJP) and Chinese (SJC) strains of S. japonicum, and between male and female S. japonicum. We found that 593, 664 and 426 probes were differentially expressed between the two geographical strains when we compared mix sexed adults, male worms and female worms. Additionally, the study revealed that 1163 male- and 1016 female-associated probes were differentially expressed in SJP whereas 1047 male- and 897 female-associated probes were differentially expressed in SJC. The study greatly expands previously published data of strain and gender-associated differential expression in S. japonicum. Further, these new data provide a stepping stone for understanding the complexities of the biology, sexual differentiation, maturation, and development of human schistosomes, signaling new approaches for identifying novel intervention and diagnostic targets against schistosomiasis.

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.

The morphology of the peripheral blood leukocytes of the tammar wallaby, Macropus eugenii, was investigated using both light and electron microscopy. In a study of 44 captive tammar wallabies, the five main families of mammalian leukocytes were identified and found to be similar to those of eutherian mammals and other marsupial species studied to date. Lymphocytes ranging in size from 5.5 to 17.5 μm were characterised by high nuclear to cytoplasmic ratio whilst monocytes were generally larger than lymphocytes at 12.4–23.4 μm and contained a single kidney shaped nucleus. Of the granulocytic populations only neutrophils and eosinophils were frequently observed and contained characteristic cytoplasmic granules. Whilst there have been a number of studies of the morphology of peripheral blood leucocytes in marsupials, a detailed description of the cells of the model marsupial, the tammar wallaby, has, to date, been lacking. This paper presents the results of the first comprehensive study of the morphology and ultrastructure of the peripheral blood cell populations of the tammar wallaby and reports that these cells are similar, though not identical, to those of eutherian mammals.

A formative assessment task was developed to improve the scientific report writing skills of university students. Students undertaking this task typically possessed varying levels of scientific literacy and were drawn from a cohort of mixed abilities. The assessment task involved the construction of a scientific report that included feedback from instructor to students before final submission of the assessment piece. After initial submission of a scientific report, the instructor developed a cohort-specific marking scheme based on the deficiencies that were evident within the class group. Using a mixture of peer and self-review against specific criteria, the students were required to resubmit an amended report. This resulted in elevated marks compared with those that would have been obtained after first submission, thus rewarding the student for the application of feedback. This technique proved to be efficient for both parties and also resulted in improvement of skills of the entire student population, regardless of the ability of the student prior to the assessment task. Using this methodology, students of varying aptitudes were able to measure their own skill improvement against tangible criteria, and enjoy a degree of learning success independent of the ranking within their group.

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.

The histology of the spleen, lymph nodes, Gut-associated lymphoid tissue (GALT) and Bronchus-associated lymphoid tissue (BALT) are described for samples collected opportunistically from healthy and mycobacteria-affected specimens of the endangered marsupial Lagorchestes hirsutus, the Rufous Hare-wallaby. The structural elements, organization and distribution of T and B lymphocytes determined by immunohistological techniques using species cross-reactive antibodies in the lymph nodes, spleen and GALT of this species demonstrated lymphoid cell distributions that were consistent with other marsupial and eutherian mammals. The tissues of animals identified as acid-fast positive displayed immunopathology consistent with the responses to intracellular bacteria displayed in some eutherian mammals and included the presence of focal lesions, giant cells in the lung and lymphoid aggregations situated adjacent to blood and airway vessels. This is the first study to describe the lymphoid tissue of this rare macropod species and the first to document the tissue bed response to mycobacteria.

Sequence data for type I interferons (IFNs) have previously only been available for birds and eutherian ('placental') mammals, but not for the other two groups of extant mammals, the marsupials and monotremes. This has left a large gap in our knowledge of the evolutionary and functional relationships of what is a complex gene family in eutherians. In this study, a PCR-based survey of type I IFN genes from a marsupial, the tammar wallaby (Macropus eugenii), and a monotreme, the short-beaked echidna (Tachyglossus aculeatus), was conducted. Along with Southern blot and phylogenetic analysis, this revealed a large number of type I IFN genes for the wallaby, rivalling that of eutherians, but relatively few type I IFN genes in the echidna. The wallaby genes include both IFNA and IFNB orthologues, indicating that the gene duplication leading to these subtypes occurred prior to the divergence of marsupials and eutherians some 130 million years ago. Results from this study support the idea that the expansion of type I IFN gene complexity in mammals coincides with a concomitant expansion in the functionality of these molecules. For example, this expansion in complexity may have, at least partially, facilitated the evolution of viviparity in marsupials and eutherians. Other evolutionary aspects of these sequences are also discussed.

Diversity in major histocompatibility complex (MHC) genes can be correlated with the level of immunological fitness of an individual or group of individuals. This study tested published primer sets designed to amplify fragments of the MHC Class II DAB and DBB genes to amplify the equivalent gene fragments in red-tailed phascogales (Phascogale calura). Seventeen genomic DNA samples extracted from phascogale muscle tissue were used to amplify the initial DAB and DBB fragments; however, only DAB PCR proved successful. The fragments were 172 bp in length between the primers and had a high level of identity to other known marsupial MHC Class II DAB gene sequences (89–98%), including those of the koala (Phascolarctos cinereus), Tasmanian devil (Sarcophilus harrisii), common brushtail possum (Trichosurus vulpecula) and several wallaby species. Multiple sequence alignment revealed limited variability of MHC Class II genes between the individuals, but eight individual sequences in total. Genomic DNA was subsequently extracted from three fresh red-tailed phascogale scat samples and DAB fragments successfully amplified. The technique will allow for red-tailed phascogales to be sampled non-invasively in the wild and to determine the level of MHC diversity among individuals in the population.

Interferon-γ (IFN-γ) is an important immune regulatory molecule that plays a significant role in internal and external modulation of the mammalian immune response to intracellular pathogens. Herein, we report the 492 nt expressed sequence for the coding domain of IFN-γ from the immune tissues of two Australian macropod marsupial species: the tammar wallaby (Macropus eugenii) and the vulnerable rufous hare-wallaby (Lagorchestes hirsutus). Both 5′ and 3′ untranslated regions and the coding domain of M. eugenii IFN-γ revealed the presence of motifs responsible for transcriptional regulation, mRNA regulation, post-translational modifications, and receptor binding in other mammals. Since diagnostic kits for mycobacterial disease commonly rely on the assessment of interferon levels, we can now use this information to develop reagents that can be applied in clinical and laboratory settings to further our understanding of marsupial responses to disease.

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.

Even before the 4G technology your smartphone uses was rolled out in earnest, telecommunications experts were dreaming of the next generation: 5G. But what 5G will do and how it will do it have remained pretty nebulous. "5G is a plethora of technologies that people are trying to bring together. What technology should be prioritized in what way?" says Thyagarajan Nandagopal, the director of the Networking Technologies and Systems program at the U.S. National Science Foundation.

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.

B cell responses and their concomitant signal transduction pathways are not well understood in marsupial mammals, despite the availability of gene expression data for key immunoglobulin genes and for elements of the CD79a/CD79b heterodimer signalling complex for two model marsupials. Broader studies of factors that influence B cell responses are still hampered by a lack of species-specific reagents and there are few reports of other factors that influence gene expression such as the potential for splice variants in BCR components, which may influence immune signalling pathways. In this study, we characterise CD79a and CD79b genes in the endangered macropod marsupial, Onychogalea fraenata (the bridled nailtail wallaby) and show that domains and residues important for the structural and functional integrity of both monomers are conserved in this species, consistent with results previously reported for the closely-related macropod, Macropus eugenii (the tammar wallaby). We extend this work to report the detection of splice variants for CD79a and CD79b in wallaby species; three CD79a isoforms and one CD79b isoform. Of these, two CD79a isoforms and the CD79b isoform have not been reported in any other mammalian species.

The complement system is a major mediator of the vertebrate immune system, which functions in both innate and specific immune responses. It comprises more than 30 proteins working to remove foreign cells by way of anaphylatoxins, opsonins or the membrane attack complex. Over the last few years, whole genome sequences of non-eutherian mammals (marsupials and a monotreme), the gray short-tailed opossum (Monodelphis domestica), tammar wallaby (Macropus eugenii), Tasmanian devil (Sarcophilus harrisii), koala (Phascolarctos cinereus) and platypus (Ornithorhynchus anatinus), have become publicly available. Using these sequences, we have identified an array of complement components in non-eutherians using online search tools and algorithms. Of 57 complement and complement-related genes investigated, we identified 46 in the gray short-tailed opossum genome, 27 in the tammar wallaby genome, 44 in the Tasmanian devil genome, 47 in the koala genome and 40 in the platypus genome. The results of this study confirm the presence of key complement components in the immune repertoire of non-eutherian mammals and provide a platform for future studies on immune protection in young marsupials.

Interleukin-1beta (IL-1β) plays a significant role in the onset and pathogenesis of inflammation in mammalian hosts. Although well characterized in a range of vertebrate species, little is known about this important cytokine in marsupial mammals. We report here the molecular cloning and characterization of IL-1β in the tammar wallaby (Macropus eugenii). M. eugenii IL-1β has an open-reading frame of 813 nucleotides, coding for a putative protein of 270 amino acids to the termination codon. The IL-1 family motif and potential caspase cleavage site (necessary for production of the mature protein) is also present in the sequence. Molecular characterization of tammar wallaby IL-1β provides fundamental information necessary to progress the study of functional immune responses in this unique group of mammals.

Very few cytokines that are important to the understanding of T helper cell function are characterized in marsupials. Expression of a 645 bp cDNA product that codes for a predicted Interleukin-4 peptide of 157 amino acids was detected in the lymph node tissues of Macropus eugenii, the tammar wallaby. Using Rapid Amplification of cDNA Ends, both 5'- and 3'-untranslated regions were identified and a polyadenylation signal and three mRNA instability motifs associated with secreted cytokine molecules were also present. The translated cDNA sequence has a putative signal peptide of 24 amino acids, a predicted secondary structure that is consistent with the short-chain alpha-helical cytokine family and 82% conservation of residues associated with the Interleukin-4 family sequence motif. Comparisons of wallaby nucleotide and predicted peptide sequences with the coding domains of other vertebrate species demonstrate the diversity within this gene family; with nucleotide and amino acid identities of 74% and 59% with opossum, 52% and 32% with human and 38% and 19% with chicken homologues respectively. Despite these differences in sequence conservation, the putative Macropus eugenii Interleukin-4 mature peptide contains conserved structural motifs and predicted receptor-binding residues that suggest that it may retain functional properties associated with this important Th2 cytokine in other mammals.

Background Cytokines are small proteins that regulate immunity in vertebrate species. Marsupial and eutherian mammals last shared a common ancestor more than 180 million years ago, so it is not surprising that attempts to isolate many key marsupial cytokines using traditional laboratory techniques have been unsuccessful. This paucity of molecular data has led some authors to suggest that the marsupial immune system is 'primitive' and not on par with the sophisticated immune system of eutherian (placental) mammals. Results The sequencing of the first marsupial genome has allowed us to identify highly divergent immune genes. We used gene prediction methods that incorporate the identification of gene location using BLAST, SYNTENY + BLAST and HMMER to identify 23 key marsupial immune genes, including IFN-γ, IL-2, IL-4, IL-6, IL-12 and IL-13, in the genome of the grey short-tailed opossum (Monodelphis domestica). Many of these genes were not predicted in the publicly available automated annotations. Conclusion The power of this approach was demonstrated by the identification of orthologous cytokines between marsupials and eutherians that share only 30% identity at the amino acid level. Furthermore, the presence of key immunological genes suggests that marsupials do indeed possess a sophisticated immune system, whose function may parallel that of eutherian mammals.

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.

Monocytes, monocyte-derived adherent cells and dendritic cells all play a role in cellular immunity. In this study, we describe the isolation of monocyte-derived adherent cells and dendritic cells from a model marsupial, the tammar wallaby, Macropus eugenii, and report that in vitro, these cells appear morphologically similar to these cells found in other mammals. The successful culture of marsupial monocyte and dendritic cells was undertaken in serum-free medium which contained lymphocyte conditioned medium as an absolute requirement. This supports the view that similar to cultured dendritic cells from other species reported to date, specific growth factors are required to promote the maturation and differentiation of these cells.

The debate about the state of the marsupial immune system has entered a new era with the recent publication of the tammar wallaby (Macropus eugenii) genome. The aim of this study was to investigate two important components of the T-cell signalling cascade in M. eugenii to determine whether there are any significant differences between the genome and the expressed gene sequences and to elucidate the putative structures. Molecular methods, predominantly RACE PCR and RT–PCR, using cDNA obtained from mRNA isolated from M. eugenii lymph node tissue, were used to determine the sequence of functional motifs in the TCRζ and ZAP-70 molecules. Structure prediction algorithms were used to determine their secondary and tertiary structures for comparison with the structures elucidated by X-ray crystallography in humans and other mammals. Differences between the genome and the expressed sequence were found in the ZAP-70 molecule. Homology modelling demonstrated that the predicted structure of the TCRζ molecule was different from those of other mammals while the structure of the ZAP-70 molecule was very similar. It is concluded that the T-cell signalling cascade in the adaptive immune system of marsupials shows similar features to that of other mammals.

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.

Background: Cytokines are small proteins that regulate immunity in vertebrate species. Marsupial and eutherian mammals last shared a common ancestor more than 180 million years ago, so it is not surprising that attempts to isolate many key marsupial cytokines using traditional laboratory techniques have been unsuccessful. This paucity of molecular data has led some authors to suggest that the marsupial immune system is \'primitive\' and not on par with the sophisticated immune system of eutherian (placental) mammals. Results: The sequencing of the first marsupial genome has allowed us to identify highly divergent immune genes. We used gene prediction methods that incorporate the identification of gene location using BLAST, SYNTENY + BLAST and HMMER to identify 23 key marsupial immune genes, including IFN-γ, IL-2, IL-4, IL-6, IL-12 and IL-13, in the genome of the grey short-tailed opossum (Monodelphis domestica). Many of these genes were not predicted in the publicly available automated annotations. Conclusion: The power of this approach was demonstrated by the identification of orthologous cytokines between marsupials and eutherians that share only 30% identity at the amino acid level. Furthermore, the presence of key immunological genes suggests that marsupials do indeed possess a sophisticated immune system, whose function may parallel that of eutherian mammals.

The changes in cellular composition of the milk of the koala (Phascolarctos cinereus) have been investigated in late lactation and after the loss of pouch and back young. During lactation, the polymorphonuclear neutrophil was the most frequently observed cell. After loss or removal of the young, macrophages and foamy epithelial cells were detected in moderate to high numbers. In one animal, mammary secretions containing lymphocytes and unidentifiable, immature mononuclear cells continued for 120 days after the loss of pouch young. These observations of cellular composition of late- and post-lactation secretions are consistent with reports from a number of eutherian mammals and also with another marsupial, the tammar wallaby (Macropus eugenii).

The cellular components of colostrum and milk of the tammar wallaby (Macropus eugenii) have been investigated over the period of oestrus, lactation and weaning. Macrophages, neutrophils, lymphocytes and other vacuolated mononuclear cells were identified. The total number and diversity of cells were higher in colostral secretions and in secretions from post-lactational mammary glands. Neutrophils were the predominant cell type in early secretions. Macrophages were more prevalent in the milk of animals that no longer had young attached to the teat. These observations are consistent with suggestions that phagocytic cells play a role in post-lactational repair of the mammary gland but also suggest that non-specific phagocytic protection plays a role in protection of the neonatal marsupial.