Physical mapping of innate immune genes, mucins and lysozymes, and other non-mucin proteins in the tammar wallaby (Macropus eugenii).

Sequencing of the tammar wallaby (Macropus eugenii) genome has the potential to be an extremely valuable resource for investigating evolutionary and developmental aspects of the mammalian immune system. However, the tammar wallaby genome has only been sequenced to a 2-fold depth and consists of small contigs, leaving many sequence gaps, many putative orthologs unpredicted and the location of genes within the genome unknown. In the case of low sequenced genomes, physical maps of genes on chromosomes can help identify specific genes if they map to conserved regions. Genes corresponding to adaptive immunity have been mapped in the tammar wallaby; however, genes corresponding to the innate immune system have not been investigated. We predict 2 types of genes important to the innate immune system, mucins and lysozymes, in the tammar wallaby and compare the predicted peptide sequences and locations of the genes with the South American opossum (Monodelphis domestica) and human. We use fluorescence in situ hybridization to physically map the genes to tammar wallaby chromosomes, demonstrating the importance of identifying and mapping genes when genomes have low sequence coverage. As mucins and lysozymes play protective roles in young animals, we also propose that their immunological role in developing marsupials warrants further investigation.

Do free-ranging common brushtail possums (Trichosurus vulpecula) play a role in the transmission of Toxoplasma gondii within a zoo environment?

To investigate the possible role of common brushtail possums (Trichosurus vulpecula) in the transmission of Toxoplasma gondii within a zoo environment, a serological survey of a free-ranging population resident within Taronga Zoo, Sydney, Australia was undertaken using the modified agglutination test (MAT). For comparison, the seroprevalence of T. gondii antibodies was also assessed in a possum population inhabiting a felid-free, non-urban woodland habitat. Six of 126 possums (4.8%) from the zoo population had antibodies to T. gondii with a MAT titre of 25 or higher, while in contrast, all of the 17 possums from woodland were seronegative. These observations suggest that possums were at a higher risk of exposure to the parasite as a consequence of co-existing with domestic, stray and captive felids associated with urbanisation. Screening of captive felids at the zoo indicated 16 of 23 individuals (67%) and all 6 species were seropositive for T. gondii, implicating them as a possible source of the parasite within the zoo setting. In addition captive, non-felid carnivores including the chimpanzee (Pan troglodytes), saltwater crocodile (Crocodylus porosus), dingo (Canis lupis) and leopard seal (Hydrurga leptonyx) were tested for the presence of T. gondii antibodies as these species predate and are a leading cause of death amongst zoo possums. In total, 5 of 23 individuals (22%) were seropositive, representing 2 of the 4 carnivorous species; the dingo and chimpanzee. These data suggest that carnivory was not a highly efficient pathway for the transmission of T. gondii and the free-ranging possum population posed minimal threat to the health of zoo animals.

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.

Culture and stimulation of tammar wallaby lymphocytes.

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

Culture and characterisation of peripheral blood monocytes and monocyte-derived adherent cells of the tammar wallaby, Macropus eugenii.

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.

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.

cDNA sequence of the lymphotoxin beta chain from a marsupial, Macropus eugenii (Tammar wallaby).

In humans and mice, the lymphotoxin (LT) complex is a heterotrimer that consists of alpha (LT-alpha) and beta (LT-beta) chains, predominantly in the ratio 1:2 (LT-alpha:LT-beta). LT-beta is a type II membrane-bound protein that anchors the complex to the surface of activated lymphocytes and, in gene targeting experiments in mice, has been shown to be crucial for normal lymphoid organogenesis. However, a similar role for LT in noneutherian mammals has not yet been established. Indeed, there has been no previous evidence for the existence of LT in noneutherian species. We have isolated, by rapid amplification of cDNA ends on mammary lymph node cDNA, the transcript coding for LT-beta from the tammar wallaby, Macropus eugenii. This constitutes the first report of an LT component from a marsupial and hence from a noneutherian mammal. The predicted amino acid sequence encoded by this transcript shares 63% and 46% sequence identity with mouse and human LT-beta, respectively.

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.

Immunology of pouch young marsupials. I. Levels of immunoglobulin transferrin and albumin in the blood and milk of euros and wallaroos (hill kangaroos: Macropus robustus, marsupialia).

The concentration of total protein, albumin, transferrin, and immunoglobulin G of adult serum, pouch young serum, milk whey and colostrum has been estimated in three species of kangaroos, Macropus robustus, Macropus rufus (= Megaleia rufa) and Macropus giganteus. No study of this kind has previously been published for any marsupial species. The three individual proteins were antigenically identical in all four kinds of fluid. Colostrum and milk whey are relatively enriched in transferrin but have low levels of immunoglobulin G. Serum concentrations of total protein, albumin and transferrin rise steadily throughout pouch life and attain adult values when the young finally leaves the pouch. Serum concentrations of immunoglobulin G are very low for the first 90-100 days of pouch life, being approximately half of those in milk whey for this period. After this the level rises rapidly and also reaches adult values when the young leaves the pouch. We suggest that in the first 90-100 days the pouch young is largely protected humorally by passive immunity acquired from the mother, and after this it increasingly makes its own responses.

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.

Further characterization of T cell receptor chains of marsupials.

cDNA clones encoding T cell receptor alpha (TCRalpha) and beta (TCRbeta) from the South American opossum, Monodelphis domestica were isolated and characterized. A single clone isolated encoding a TCRalpha chain was full length, containing the complete V (variable), J (joining) and C (constant) regions. Three partial cDNA clones were isolated for TCRbeta which contained complete C sequences. Phylogenetic analysis of the TCR Valpha revealed that the M. domestica sequence and a sequence from the Australian brushtail possum, Trichosurus vulpecula, belong to separate Valpha families and intersperse with sequences from eutherian mammals. Similar to results described for marsupial and eutherian light chains, diversity at the V region of the TCR is ancient and maintained. In contrast phylogenetic analysis of the TCR Calpha and Cbeta sequences from M. domestica, T. vulpecula, and other vertebrates revealed that the marsupial TCR C grouped together forming a sister group to eutherian mammals.

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.

Development of the lymphoid tissues of the tammar wallaby Macropus eugenii.

A study has been made of the development of four lymphoid tissues from birth to maturity in the tammar wallaby Macropus eugenii--the cervical and thoracic thymus, lymph nodes and gut-associated lymphoid tissue (GALT). The development of these tissues in the tammar wallaby is similar to that in two other marsupials, the quokka Setonix brachyurus and the Virginian opossum Didelphis virginiana. Lymphocytes were first detected in the cervical thymus of the tammar at Day 2 post partum and in the thoracic thymus at Day 6. They were subsequently detected in lymph nodes at Day 4 and in the spleen by Day 12 but were not apparent in the GALT until around Day 90 post partum. By Day 21, the cervical thymus had developed distinct areas of cortex and medulla and Hassall's corpuscles were apparent. The maturation of other tissues followed with Hassall's corpuscles in the thoracic thymus by Day 30 and nodules and germinal centres in the lymph nodes by Day 90. Measurement of immunoglobulin G concentrations in the serum of young animals indicated a rise in titre around Day 90 post partum, correlating with the apparent maturation of the lymphoid tissues.

Development of the blood-forming tissues of the tammar wallaby Macropus eugenii.

The development of the haematopoietic tissues of the tammar wallaby Macropus eugenii follows a similar pattern to that observed in eutherian and other metatherian mammals. At birth, the liver appears to be the only site of haematopoiesis with significant numbers of neutrophils and stem cells present in the circulation. By Day 3, the spleen shows limited haematopoietic activity and by Day 12 contains areas of erythroid and myeloid cells. At two weeks after birth, the haematopoetic activity in the liver declines and small areas of haematopoiesis are apparent in the bone marrow. By the end of the first month, the bone marrow appears to be the major site of haematopoiesis.

Immunoglobulin G levels in fetal and newborn tammar wallabies (Macropus eugenii).

Immunoglobulin G (IgG) was measured in fetal, neonatal and colostral samples from the tammar wallaby (Macropus eugenii) in order to study the possibility of passively acquired immunity. Samples were obtained from young at a known stage of gestation and at known times (to the minute) after birth. IgG was present (in increasing levels of concentration) in fetal serum, neonatal serum and colostrum. Since the fetus and neonate are probably unable to make immunoglobulin (Ig), it is hypothesized that transplacental and trans-gut transmission takes place from mother to offspring. The vascular yolk sac placenta has a high concentration of IgG, and is the most likely route of transmission from mother to young. Some observations were made of IgA which was found only in colostrum. No Ig of either kind was found in yolk sac fluid.

Gestational length in the koala, Phascolarctos cinereus.

We report a possible case of extended gestation in the koala, Phascolarctos cinereus. Birth of a pouch young was first observed 127 days after the removal of the male from a multi-female colony at Taronga Zoo. No other males were present at that time or had access to the facility. Head measurements and other growth data collected at the time of detection and over the period of pouch life indicates the time from removal of the male and the date of birth to be between 50 and 77 days. DNA fingerprinting using microsatellite loci unambiguously assigned paternity of the pouch young to this male. These observations suggest either an extended period of gestation of at least 50 days, or activation of a dormant blastocyst from the previous breeding season, as the female entered the period of seasonal oestrus.

cDNA cloning of lymphotoxin alpha (LT-alpha) from a marsupial, Macropus eugenii.

Lymphotoxin (LT) is a proinflammatory cytokine with a broad spectrum of immunological activities. While the 'classic' form of the molecule is a secreted homotrimer, now referred to in the literature as LT-alpha3, it has more recently been recognised that a membrane-bound form of LT exists on activated T lymphocytes and that this represents a complex between LT-alpha and a closely related type II membrane protein, LT-beta. Together with another related cytokine, tumour necrosis factor alpha (TNF-alpha), these molecules have been extremely well studied in eutherian mammals but not in any other group. Marsupials represent a distinct branch in mammalian evolution to that of eutherians, the two groups having diverged more than 100 million years ago. We report here for the first time, the cDNA cloning of LT-alpha from a marsupial, Macropus eugenii (tammar wallaby). This sequence was found to be relatively conserved when compared to orthologous sequences from eutherian mammals, sharing an average sequence identity of 70.4% at the nucleotide level and 71.7% at the deduced amino acid level.

Canine coronavirus in Australian dogs.

OBJECTIVE: To estimate the frequency of serum antibodies (IgG and IgM) to canine coronavirus (CCV) in the Australian dog population and evaluate the role of CCV as a causative agent of gastroenteritis. DESIGN: A serological survey of antibodies to CCV among different dog populations. PROCEDURE: The development and characterisation of an indirect ELISA for the detection of antibodies (IgG and IgM) to CCV was undertaken. Sera collected from both diarrhoeal and non-diarrhoeal dogs from various populations throughout Australia were tested for these antibodies to CCV. RESULTS: Serum samples (1396) collected from 1984 to 1998 were tested for the presence of IgG antibodies to CCV. Samples were divided into two categories on the basis of the number of dogs housed together. The groups were either an open population containing dogs housed as groups of three or less, or kennel populations. Sera from 15.8% of the open population and 40.8% of kennelled dogs were positive for CCV antibodies. The prevalence of antibodies varied from zero to 76% in kennelled dogs. About 23% of 128 dogs positive for IgG antibodies to CCV were also positive for IgM antibodies to CCV, indicating recent CCV infection. Of those dogs that were presented with clinical signs of gastroenteritis such as diarrhoea and vomiting (n = 29), 85% were positive in the IgM ELISA and 85.7% in the IgG ELISA for antibodies to CCV. In comparison, for those dogs presented without any history of gastroenteritis only 15% were positive for IgM and 30% positive for IgG. CONCLUSION: Serological evidence indicates that infection with CCV in dogs is widespread throughout the Australian mainland. The prevalence of antibodies varies greatly among different populations, with an average of 40.8% positive in kennelled populations and 15.8% in the open population.