Limitations in the isolation and stimulation of splenic mononuclear cells in a dasyurid marsupial, Phascogale calura.

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.

Sarcoptic mange in wombats-A review and future research directions.

Sarcoptic mange is caused by the mite Sarcoptes scabiei and has recently been recognized as an emerging infectious disease of wildlife worldwide. The mite is one of the main causes of population decline in southern hairy-nosed (Lasiorhinus latifrons) and bare-nosed wombats (Vombatus ursinus). This review focuses on Sarcoptes scabiei infestations in wombats and provides insights into why the disease may be so prevalent in wombats. Current treatment practices and trials conducted in the field to reduce the incidence of sarcoptic mange in wombats are described and critically reviewed. Current and potential future avenues of research are discussed.

The common gamma chain cytokine interleukin-21 is expressed by activated lymphocytes from two macropod marsupials, Macropus eugenii and Onychogalea fraenata.

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.

Scrub-itch mite infestation in the endangered bridled nailtail wallaby.

Skin lesions on the ears and inguinal and axillary regions of a number of adult animals within a captive population of the endangered bridled nailtail wallaby (Onychogalea fraenata) were associated with the trombiculid mite, Eutrombicula hirsti. The local inflammatory response of these Australian marsupials is described.

Characterisation of the CD5 cDNA sequence from the tammar wallaby (Macropus eugenii).

CD5 has previously been identified in marsupial tissues using anti-human CD5. However, despite the known cross-reactivity of the antibody in marsupial tissues, the cDNA sequence has not previously been characterised in any marsupial. This study has identified the CD5 gene in the opossum genome database and has characterised the CD5 cDNA sequence from the tammar wallaby. Both marsupial CD5 sequences have a high level of sequence identity to known eutherian CD5 sequences, are cysteine-rich and have identical structural motifs to their eutherian homologs. CD5 transcripts were strongly expressed in adult tammar wallaby spleen, mammary node and blood, and expressed at a lower level in liver, kidney and heart tissues. Characterisation of CD5 in marsupials allowed a comparison to the epitope sequence of anti-human CD5 and showed a high level of sequence identity.

Isolation of the mite Myocoptes musculinus Koch from the Spinifex Hopping mouse (Notomys alexis).

This paper reports on the isolation and identification of the fur-clasping mite, Myocoptes musculinus, from the faeces of the Spinifex Hopping mouse (Notomys alexis). This investigation adds to the sparse records of ectoparasites collected from native Australian murids.

Screening and genetic diagnosis of haemoglobinopathies.

The haemoglobin disorders are a group of autosomal recessive disorders characterized by either the reduced synthesis of one or more normal globin chains (the thalassaemias), the synthesis of a structurally abnormal globin chain (the haemoglobin variants) or in a few cases by both phenotypes (the reduced synthesis of a Hb variant, e.g. Hb E). They are the commonest single-gene disorders known and approximately 1000 different mutant alleles have now been characterized at the molecular level. The mutations are regionally specific, with each country having its own unique spectrum of abnormal haemoglobins and thalassaemia mutations, and can occur at high gene frequencies in some ethnic groups 1. Although haemoglobinopathy mutations are rarely found in individuals of North European origin, the number of immigrants in the North European countries is steadily increasing and the variety of their ethnic origins poses a problem for screening and accurate diagnosis.

The immune tissues of the endangered red-tailed phascogale (Phascogale calura).

The lymphoid tissues of the red-tailed phascogale (Phascogale calura) were examined using histological and immunohistochemical techniques. The distribution of immune cells in the tissue beds was documented using antibodies to surface markers CD3 and an MHC Class II antigen (equivalent to HLA DRII). Spleen, gut-associated lymphoid tissues (GALT), lung, bronchus-associated lymphoid tissue (BALT) and liver were examined. The spleen had defined areas of red and white pulp, with follicles containing tingible-bodied macrophages. Anti-CD3 and anti-HLA DRII antibodies revealed the presence of T cells in areas of white pulp and around the peri-arterial lymphatic sheaths. GALT and BALT were detected and appeared as scattered areas of lymphocytes in the tissues beds. This is the first study to report on the lymphoid tissues of this endangered species of marsupial and the first report of the capacity of anti-human antibodies to a surface MHC molecule to react with Dasyurid cells.

The appearance and distribution of mature T and B cells in the developing immune tissues of the stripe-faced dunnart (Sminthopsis macroura).

This paper describes the initial appearance and distribution of mature T and B cells in the developing immune tissues of the stripe-faced dunnart (Sminthopsis macroura) based on the use of species cross-reactive antibodies to the lymphocyte cell surface markers CD3, CD5 and CD79b. At birth no mature T or B cells were detected in the liver or bone marrow using anti-CD3, anti-CD5 or anti-CD79b antibodies. T cells were detected in the thymus with anti-CD3 by day 12 and anti-CD5 by day 50 postpartum, and T cells in the spleen were detected by day 43 and day 80 postpartum using anti-CD3 and anti-CD5, respectively. B cells were observed in the dunnart spleen by 43 days after birth. CD3- and CD79b-positive cells were detected in the lymph nodes by 50 days and CD5 by day 15 after birth, and in the gut-associated lymphoid tissues by day 50 and anti-CD5 by day 57 postpartum. The development and distribution of T and B cells in the immune tissues of dunnart pouch young is similar to that described in other marsupial species. Low numbers or absence of mature lymphocytes in immune tissues of early pouch young dunnarts further support the proposition that young marsupials are reliant on non-specific defence strategies and/or maternal strategies for a significant period of their time of development in the pouch.