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.

A developmental investigation of the liver, bone marrow and spleen of the stripe-faced dunnart (Sminthopsis macroura).

The development of the liver, bone marrow and spleen have been investigated in the stripe-faced dunnart. At birth, the liver was undergoing haematopoiesis but the level declined rapidly and by day 50 after birth the liver was histologically mature. Both the bone marrow and spleen were non-haematopoietic at birth but initiated haematopoiesis shortly thereafter. Bone marrow was initially detected at day 11 postpartum. By 57 days after birth, adipocytes had infiltrated the marrow and were abundant by day 60 after birth. Mitotic cells were observed in remaining areas of marrow until at least 170 days postpartum. The spleen at birth was undifferentiated, with trabeculae appearing by day 42. Red and white pulp areas became apparent by day 43 and were well defined by day 57 after birth. In summary, the pattern of the development of the liver, bone marrow and spleen in the stripe-faced dunnart were similar to that observed in eutherians and other metatherians studied to date.

The detection of mature T- and B-cells during development of the lymphoid tissues of the tammar wallaby (Macropus eugenii).

The distribution of T- and B-cells in the developing lymphoid and immunohaematopoietic tissues of the tammar wallaby were investigated using antibodies to the mature cell surface markers, CD3, CD5 and CD79b. In the thymus, CD3- and CD5-positive T-cells were first observed at day 12 postpartum whilst rare B-cells were first detected at day 23. Both T- and B-lymphocytes were first stained on day 21 postpartum in the spleen and day 24 in lymph nodes. In one sample from a 7-day-old animal, rare CD79b-positive (CD79b+) lymphocytes were observed in the gut-associated lymphoid tissues. However, CD3+ cells were not apparent until day 12 and CD5+ cells were not detected until day 74 postpartum. No lymphocytes were detected in liver or bone marrow samples and no bronchus-associated lymphoid tissues were observed. The pattern of development and the distribution of T- and B-cells in the lymphoid and immunohaematopoietic tissues were similar to those observed in eutherian mammals and in limited studies of other metatherians. However, the detection of apparently mature T- and B-cells in the thymus and gut-associated lymphoid tissues (GALT) at the same postnatal age highlights the need for a more substantial study of the development of GALT. This is, at present, limited by availability of marsupial-specific antibodies.

A histological investigation of the lymphoid and immunohaematopoietic tissues of the adult stripe-faced dunnart (Sminthopsis macroura).

This is the first published description of the lymphoid and immunohaematopoietic tissues of an Australian polyprotodont, the stripe-faced dunnart, Sminthopsis macroura and the first account of bronchus-associated lymphoid tissue (BALT) in a metatherian. Histologically, the tissue beds are similar in appearance to those reported in other adult eutherian and metatherian mammals. The liver and bone marrow were mature and virtually no haematopoietic activity was observed. The thymus had undergone involution but retained some lymphocytes. The spleen was similar to that observed in other metatherians containing areas of red and white pulp separated by a marginal zone. Lymph nodes, except for a pair in the posterior abdomen, were difficult to locate but were similar to those observed previously in other adult metatherians. Peyer's patches were present; however, they lacked dome regions and sometimes had villi above them. BALT appeared to be both compartmentalised and non-compartmentalised in the adult stripe-faced dunnart.

The lymphoid and immunohaematopoietic tissues of the embryonic brushtail possum ( Trichosurus vulpecula).

The lymphoid and immunohaematopoietic tissues of the embryonic and full-term brushtail possums was investigated histologically and immunohistochemically using antibodies to the T- and B-cell markers, CD3, CD5, CD79a and CD79b. No clearly defined thymus, bone marrow, spleen, lymph nodes, gut-associated lymphoid tissues or bronchus-associated lymphoid tissues were observed histologically. The liver was haematopoietic and contained erythrocytic and granulocytic precursors. No mature lymphocytes were observed histologically or detected using antibodies to T- and B-cell markers in any of the tissues. These results are consistent with other studies of the early postnatal tissues of other marsupials and support the proposition that neonatal marsupials are substantially reliant on maternal immunological protection at the time of birth and for a significant period of pouch life.

Screening and genetic diagnosis of haemoglobin disorders.

The inherited haemoglobinopathies are large group of disorders that include the thalassaemias and sickle cell disease. Carrier detection methods must be able to detect alpha-, beta- and deltabeta-thalassaemias, HPFH disorders and haemoglobin variants. Carrier diagnosis involves the accurate measurement of MCH, MCV, Hb A(2) and Hb F values in combination with an understanding of the haematological characteristics of the different types of thalassaemia genes and their interactions. The majority of the common thalassaemia mutations and abnormal haemoglobins can be identified by PCR-based techniques. The main applications of molecular analysis for carrier diagnosis are: the analysis of alpha-thalassaemia mutations by gap-PCR to discriminate between heterozygous alpha-thalassaemia and homozygous alpha-thalassaemia; the identification of beta-thalassaemia mutations for patients requiring prenatal diagnosis and for the prediction of the severity of the clinical phenotype of homozygous beta-thalassaemia; to discriminate between deltabeta-thalassaemia and HPFH deletions by gap-PCR.

Development of lymphoid tissues of the stripe-faced dunnart (Sminthopsis macroura).

The development of the lymphoid tissues of a model marsupial, the stripe-faced dunnart, has been described from birth to weaning, a period of 2.5 months. At birth the lymphoid tissues, including the thymus, lymph nodes and mucosa-associated lymphoid tissues, were undeveloped. A thoracic thymus consisting primarily of stromal tissue was observed by day 4 after birth but by day 12, lymphocytes were observed in the thymus and some cortico-medullary differentiation was apparent. Lymph nodes were histologically mature by day 31, the earliest day investigated for this tissue. In gut tissue, lymphoid follicles were first observed by day 57 post-partum. No bronchus-associated lymphoid tissue was observed in any lung samples. The thymus, lymph nodes and gut-associated lymphoid tissues were all distinguishable before weaning (day 70) but not all were histologically mature. The sequence of development of the lymphoid tissues in the stripe-faced dunnart was similar to those observed in other marsupial species.

The gut-associated lymphoid tissues of the northern brown bandicoot (Isoodon macrourus).

The gut associated lymphoid tissues (GALT) of a juvenile bandicoot has been examined using histological and immunohistochemical techniques. The mesenteric lymph nodes were hyperfollicular and had well defined paracortical and medullary areas. Lymphocytes were densely packed throughout the cortex and paracortex and the mantles of the follicles. The GALT contained two distinct areas of tissue organisation. One consisted of large areas of aggregated follicles, whilst the other consisted of more linearly distributed follicles. The distribution of T and B cells in the tissue beds was documented using antibodies to surface markers CD3, CD5 and CD79b. T-cells were present in high numbers in the cortical region of the lymph node, whilst B-cells were predominant in the mantle of the follicles. Dispersed CD3 positive T-cells were abundant in the villi lacteals and present in high numbers in follicular areas of gut. CD79b positive B-cells were not observed in the lacteals but were abundant in the mantles of follicles. This is similar to that observed in other metatherians.

Molecular characterisation of the tammar wallaby (Macropus eugenii) CD3 epsilon chain cDNA.

The cDNA encoding the epsilon chain of the tammar wallaby CD3 complex (CD3epsilon) was isolated by PCR. This is the first CD3 component to be cloned in a marsupial. The tammar wallaby cDNA coding region was 61.7 and 63.0% identical to the human and mouse cDNA coding sequences, respectively. Similarly, the predicted amino acid sequence was 56.5 and 52.9% identical to the human and mouse sequences. When compared with other known CD3epsilon peptide sequences, the most conserved region of the tammar wallaby CD3epsilon chain peptide was the cytoplasmic domain and the least conserved was the extracellular portion. Phylogenetic reconstruction based on the deduced amino acid sequence placed the tammar wallaby sequence in its expected position outside of all the eutherian mammals.

Histology and immunohistochemistry of the gut-associated lymphoid tissue of the eastern grey kangaroo, Macropus giganteus.

Mesenteric lymph nodes and gut-associated lymphoid tissue (GALT) from juvenile eastern grey kangaroos were investigated. The mesenteric nodes had a similar structure to that described for eutherian mammals. They contained distinct regions of medulla and cortex, with prominent follicles and germinal centres. Gut associated lymphoid tissue consisted of areas of submucosal follicles. These varied from areas of densely packed lymphocytes with darkly staining, prominent coronas to areas with no defined follicles. The distribution of T cells in these tissues was documented by use of species-crossreactive antibodies to the surface markers CD3 and CD5; B cells were identified by antibodies to CD79b. Within the lymph nodes T cells were located mainly in the paracortex and cortex, with limited numbers observed in the follicles; B cells were located on the marginal zone of the follicles. In GALT, T cells were located in the peripheral regions of the germinal centres of secondary follicles, while B cells were abundant in primary follicles. These observations are consistent with those made in a range of other marsupials (metatherian) and eutherian mammals and are indicative of the capacity to respond to antigens entering via the mouth.