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.

Linkage analysis of nondeletion hereditary persistence of fetal hemoglobin.

Nondeletion forms of hereditary persistence of fetal hemoglobin may result from regulatory disorders of globin gene expression. The defects in two such conditions were localized by demonstrating a tight genetic linkage between the disorders and polymorphic restriction endonuclease sites within the beta-like globin gene complex. In one instance, the defect probably occurred outside the region of DNA between the epsilon- and beta-globin genes.

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.

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.

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.

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 the immune system and immunological protection in marsupial pouch young.

At birth the tissues of marsupial immune system are underdeveloped. The young animal is not immunocompetent. Histological and immunohistochemical studies of pouch young epithelial tissues provide a clear picture of tissue development but the timing of onset of immunocompetence awaits definition. The survival of the neonatal marsupial in a microbially rich environment is dependent on maternal strategies, including immunoglobulin transfer via milk and, in some species, prenatally via the yolk sac placenta. It is also likely that pouch secretions play a role. This review summarizes our current knowledge of the pathway of immunological development in marsupials and the protection and threats afforded by the pouch environment.

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.

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.

Prevalence and molecular basis of alpha thalassaemia in British South Asians.

OBJECTIVES: To determine the prevalence and molecular basis of alpha thalassaemia in the British South Asian population, and the implications for genetic screening for haemoglobin disorders. METHODS: 397 South Asian Subjects were screened for haemoglobinopathy with measurement of bull blood count, haemoglobin electrophoresis, haemoglobin A2 quantification, and plasma ferritin determination. alpha Globin gene mapping was successfully performed on 266 stored buffy coat samples using a Southern blot technique after hybridisation with Bg/II and BamHI. RESULTS: Of the 266 subjects in whom gene mapping was performed, 28 had a single alpha+ thalassaemia deletion and one was homozygous for this deletion (gene frequency 0.056). Half of the heterozygotes had normal mean cell haemoglobin (MCH) values. A further 16 subjects had probable non-deletional alpha+ thalassaemia. None had alpha 0 thalassaemia. CONCLUSIONS: alpha Thalassaemia is by far the commonest haemoglobinopathy in British South Asian subjects, but is not a cause of serious genetic risk. Screening and counselling should focus on subjects with more marked red cell hypochromia (MCH < 25 pg), thus concentrating resources on subjects of different ethnic origins who may carry the alpha 0 thalassaemia gene.

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.

The effect of oestrus and the presence of pouch young on aerobic bacteria isolated from the pouch of the tammar wallaby, Macropus eugenii.

Qualitative changes in the culturable, aerobic bacterial flora isolated from the tammar pouch have been documented over the period leading up to oestrus, at the time of anticipated birth and in absence and presence of pouch young of varying ages. In a group of 12 animals studied thirty species of aerobic bacteria were isolated. Twenty five species were found in pouches with no pouch young, 9 in pouches with young less than 3 weeks of age and 9 in pouches containing older animals. Gram positive organisms including Corynebacterium spp., Micrococcus spp. and Staphylococcus spp. were the most frequent isolates, regardless of reproductive status. Whilst Gram-negative rods Klebsiella pneumoniae, Pseudomonas aeruginosa, Enterobacter aerogenes, and Escherichia coli were found in pouches with and without pouch young, but not in pouches close to oestrus, at the time of birth or in the presence of very young animals (< 6 days). While pouches without pouch young displayed the greatest diversity of bacterial species, there were still significant numbers of bacterial species in those containing pouch young. Results suggest that the microbial population of the tammar pouch does not seriously compromise the well-being of the young animal during crucial stages of immunological development. The data are consistent with observations on the microflora of the quokka pouch but not with data from the koala.

Hemoglobinopathies : community clues to mutation detection.

The hemoglobinopathies are a diverse group of inherited recessive disorders consisting of the structural hemoglobin variants and the thalassemias. They can occur at very high carrier frequencies in the malarious regions of the world and are regionally specific, with each population having a unique combination of structural variants and thalassemia mutations. Therefore, knowledge of the ethnic origin of a patient is usually essential for the quick identification of the underlying molecular defect(s) in the globin genes.

Detection of beta-thalassaemia mutations in eastern Indian population by polymerase chain reaction.

We report here the application of a non-radioactive amplification refractory mutation system (ARMS) for the direct detection of beta-thalassaemia using polymerase chain reaction. Seven beta-thalassaemia mutations accounting for 89 per cent (71 of 80) of the alleles in eastern Indian population have been identified and majority (67.5%) were due to IVS-1 nt 5 (G-C) mutation. Interestingly, 9 cases did not reveal any of the 17 common mutations reported so far from Indian population.