ANTIBODY RESPONSE TO EPSILON TOXIN OF CLOSTRIDIUM PERFRINGENS IN CAPTIVE ADULT SPRINGBOK (ANTIDORCAS MARSUPIALIS), IMPALA (AEPYCEROS MELAMPUS), ALPACA (VICUGNA PACOS), AND RED-NECKED WALLABY (MACROPUS RUFOGRISEUS) OVER A YEAR.

Enterotoxemia is an important issue in various zoological taxa. In this study, serologic responses over a 1-yr period after vaccination with a multivalent clostridial vaccine were evaluated in 10 adult springboks (Antidorcas marsupialis), 12 impalas (Aepyceros melampus), seven alpacas (Vicugna pacos), and five red-necked wallabies (Macropus rufogriseus). Antibody production to the Clostridium perfringens type D epsilon toxin component of the vaccine was measured using an indirect enzyme-linked immunosorbent assay and determined as the percentage of inhibition (% inhib). Initial % inhib was (0.01-18.9)%. All animals received initial vaccination with a booster vaccine 4 weeks apart. Serum samples were collected at T0 (nonvaccinated), 15, 30, 60, 180, and 360 days postvaccination (dpv) for analysis. The vaccine induced a high antibody response that peaked at 15, 30, and 60 dpv in springboks, 30 and 60 dpv in impalas (P < 0.01), and 60 dpv in alpacas and wallabies (P < 0.01). The booster vaccine was followed by a high antibody response, which slowly decreased with time. The antibody response was significantly higher at 360 dpv than at T0 in wallabies and alpacas (P < 0.01). In impalas and springboks, it appeared that a booster every 6 mo might be required to maintain an antibody response above baseline (P < 0.01). Because no challenge studies were performed, it is unknown whether the measured humoral immune responses would have been protective. Further research is warranted to investigate protective effects of antibodies to inoculation challenge in nondomestic species.

Further characterisation of cytokines in macropod marsupials: IL-10 and IL-10Δ3.

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 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.

Marsupial and monotreme serum immunoglobulin binding by proteins A, G and L and anti-kangaroo antibody.

Serological studies are often conducted to examine exposure to infectious agents in wildlife populations. However, specific immunological reagents for wildlife species are seldom available and can limit the study of infectious diseases in these animals. This study examined the ability of four commercially available immunoglobulin-binding reagents to bind serum immunoglobulins from 17 species within the Marsupialia and Monotremata. Serum samples were assessed for binding, using immunoblots and ELISAs (Enzyme-linked immunosorbent assays), to three microbially-derived proteins - staphylococcal protein A, streptococcal protein G and peptostreptococcal protein L. Additionally, an anti-kangaroo antibody was included for comparison. The inter- and intra-familial binding patterns of the reagents to serum immunoglobulins varied and evolutionary distance between animal species was not an accurate predictor of the ability of reagents to bind immunoglobulins. Results from this study can be used to inform the selection of appropriate immunological reagents in future serological studies in these clades.

Characterisation of major histocompatibility complex class I genes at the fetal-maternal interface of marsupials.

Major histocompatibility complex class I molecules (MHC-I) are expressed at the cell surface and are responsible for the presentation of self and non-self antigen repertoires to the immune system. Eutherian mammals express both classical and non-classical MHC-I molecules in the placenta, the latter of which are thought to modulate the maternal immune response during pregnancy. Marsupials last shared a common ancestor with eutherian mammals such as humans and mice over 160 million years ago. Since, like eutherians, they have an intra-uterine development dependent on a placenta, albeit a short-lived and less invasive one, they provide an opportunity to investigate the evolution of MHC-I expression at the fetal-maternal interface. We have characterised MHC-I mRNA expression in reproductive tissues of the tammar wallaby (Macropus eugenii) from the time of placental attachment to day 25 of the 26.5 day pregnancy. Putative classical MHC-I genes were expressed in the choriovitelline placenta, fetus, and gravid endometrium throughout the whole of this period. The MHC-I classical sequences were phylogenetically most similar to the Maeu-UC (50/100 clones) and Maeu-UA genes (7/100 clones). Expression of three non-classical MHC-I genes (Maeu-UD, Maeu-UK and Maeu-UM) were also present in placental samples. The results suggest that expression of classical and non-classical MHC-I genes in extant marsupial and eutherian mammals may have been necessary for the evolution of the ancestral therian placenta and survival of the mammalian fetus at the maternal-fetal interface.

Isoforms of the CD79 signal transduction component of the macropod B-cell receptor.

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.

Molecular characterisation and expression of interleukin-6 and interleukin-6Δ2 in the Tammar wallaby (Macropus eugenii).

The pro-inflammatory cytokine, Interleukin-6 (IL-6), has not yet been fully characterised in the model macropod, Macropus eugenii, due to incomplete sequence information in publically available genome databases. Using a Rapid Amplification of cDNA Ends strategy we have confirmed the expression and complete nucleotide sequence for this molecule in lymph node tissue and activated leukocytes. Structural conservation of the mature wallaby IL-6 molecule was high when compared with human IL-6, although there was only 34% amino acid sequence identity with the human IL-6 peptide, consistent with reports of the evolutionary divergence of this cytokine. We also report the discovery of MeIL-6Δ2, a splice variant missing exon 2, which directly translates to a truncated non-functional peptide, but which may also code for an alternative peptide that is translated downstream of the canonical IL-6 start site. This putative gene product is predicted to maintain some, if not all, of the functions of macropod IL-6 and is the first IL-6 isoform reported outside of eutherian mammals.

Immunohistochemical localization of T-lymphocyte subsets in the developing lymphoid tissues of the tammar wallaby (Macropus eugenii).

Research into marsupial adaptive immunity during ontogeny has been hampered by the lack of antibodies that react to marsupial immunological cell populations. In this study, newly synthesised polyclonal antibodies to the T cell marker, CD8, have been developed and used to investigate the ontogeny and distribution of this T cell population in the tammar wallaby. Immunohistochemical analysis indicated that the distribution of the CD8 lymphocytes in the lymphoid tissues of tammar neonates during the first 144 days of pouch life was similar to that of the eutherian mammals. However, CD8α(+) lymphocytes were observed in the intestines of tammar neonates prior to their first appearance in the cervical thymus, an observation that has not been found in eutherians. A dual labelling immunohistochemical approach was used for the indirect demonstration of CD4 and enabled the simultaneous detection in the tammar wallaby tissues of the two major T-lymphocyte populations, CD4 and CD8 that are associated with adaptive immunity. As in eutherian mammals, CD4(+) cells were the predominant T cell lymphocyte subset observed in the spleen while in the nodal tissues, an age-related decrease in the CD4(+)/CD8(+) ratio was noted. These antibodies provide a new immunological tool to study the role of T cell subsets in marsupial immunity and disease pathogenesis studies.

Antigenic compartmentation of the cerebellar cortex in an Australian marsupial, the tammar wallaby Macropus eugenii.

The mammalian cerebellar cortex is apparently uniform in composition, but a complex heterogeneous pattern can be revealed by using biochemical markers such as zebrin II/aldolase C, which is expressed by a subset of Purkinje cells that form a highly reproducible array of transverse zones and parasagittal stripes. The architecture revealed by zebrin II expression is conserved among many taxa of birds and mammals. In this report zebrin II immunohistochemistry has been used in both section and whole-mount preparations to analyze the cerebellar architecture of the Australian tammar wallaby (Macropus eugenii). The gross appearance of the wallaby cerebellum is remarkable, with unusually elaborate cerebellar lobules with multiple sublobules and fissures. However, despite the morphological complexity, the underlying zone and stripe architecture is conserved and the typical mammalian organization is present.

WFDC2 is differentially expressed in the mammary gland of the tammar wallaby and provides immune protection to the mammary gland and the developing pouch young.

WAP four disulfide core domain 2 (WFDC2) is a four disulfide core (4-DSC) protein secreted in the milk of the tammar wallaby. It is comprised of two 4-DSC domains assigned domain III at the NH2-terminal end and domain II at the COOH-terminal end. The WFDC2 gene was expressed only during pregnancy, early lactation, towards the end of lactation and involution. The WFDC2 protein showed antibacterial activity against Staphylococcus aureus, Salmonella enterica and Pseudomonas aeruginosa and this activity resided with domain II. There was no antibacterial activity detected against Enterococcus faecalis. The observed expression pattern of tammar WFDC2 and its antibacterial activity suggests a role to either reduce mastitis in the mammary gland caused by S. aureus or to protect the gut of the young at a time when it is not immune-competent. The latter effect could be achieved without disturbing the balance of commensal gut flora such as E. faecalis.

Tammar wallaby mammary cathelicidins are differentially expressed during lactation and exhibit antimicrobial and cell proliferative activity.

Cathelicidins secreted in milk may be central to autocrine feedback in the mammary gland for optimal development in addition to conferring innate immunity to both the mammary gland and the neonate. This study exploits the unique reproductive strategy of the tammar wallaby (Macropus eugenii) model to analyse differential splicing of cathelicidin genes and to evaluate the bactericidal activity and effect of the protein on mammary epithelial cell proliferation. Two linear peptides, Con73 and Con218, derived from the heterogeneous carboxyl end of cathelicidin transcripts, MaeuCath1 and MaeuCath7 respectively, were evaluated for antimicrobial activity. Both Con73 and Con218 significantly inhibited the growth of Staphylococcus aureus, Pseudomonas aureginosa, Enterococcus faecalis and Salmonella enterica. In addition both MaeuCath1 and MaeuCath7 stimulated proliferation of primary tammar wallaby mammary epithelial cells (WallMEC). Lactation-phase specific alternate spliced transcripts were determined for MaeuCath1 showing utilisation of both antimicrobial and proliferative functions are required by the mammary gland and the suckled young. The study has shown for the first time that temporal regulation of milk cathelicidins may be crucial in antimicrobial protection of the mammary gland and suckled young and mammary cell proliferation.

The tammar wallaby major histocompatibility complex shows evidence of past genomic instability.

BACKGROUND: The major histocompatibility complex (MHC) is a group of genes with a variety of roles in the innate and adaptive immune responses. MHC genes form a genetically linked cluster in eutherian mammals, an organization that is thought to confer functional and evolutionary advantages to the immune system. The tammar wallaby (Macropus eugenii), an Australian marsupial, provides a unique model for understanding MHC gene evolution, as many of its antigen presenting genes are not linked to the MHC, but are scattered around the genome. RESULTS: Here we describe the 'core' tammar wallaby MHC region on chromosome 2q by ordering and sequencing 33 BAC clones, covering over 4.5 MB and containing 129 genes. When compared to the MHC region of the South American opossum, eutherian mammals and non-mammals, the wallaby MHC has a novel gene organization. The wallaby has undergone an expansion of MHC class II genes, which are separated into two clusters by the class III genes. The antigen processing genes have undergone duplication, resulting in two copies of TAP1 and three copies of TAP2. Notably, Kangaroo Endogenous Retroviral Elements are present within the region and may have contributed to the genomic instability. CONCLUSIONS: The wallaby MHC has been extensively remodeled since the American and Australian marsupials last shared a common ancestor. The instability is characterized by the movement of antigen presenting genes away from the core MHC, most likely via the presence and activity of retroviral elements. We propose that the movement of class II genes away from the ancestral class II region has allowed this gene family to expand and diversify in the wallaby. The duplication of TAP genes in the wallaby MHC makes this species a unique model organism for studying the relationship between MHC gene organization and function.

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.

Sub-cellular localisation of alkaline phosphatase activity in the cytoplasm of tammar wallaby (Macropus eugenii) neutrophils and eosinophils.

Alkaline phosphatase (ALP) has been used in studies of neutrophil morphology and function as a marker for identifying different granule populations. In human neutrophils, ALP is found within secretory vesicles, a rapidly mobilisable vesicle population important for upregulating membrane receptors during early activation. Intra-cellular ALP activity in the heterophils of rabbits and guinea pigs, in contrast, is found only in secondary granules. The neutrophils and eosinophils of tammar wallabies (Macropus eugenii) have previously been reported to contain large amounts of ALP activity when stained using routine cytochemical techniques. To define the subcellular location of ALP in this species, cell suspensions were examined using cerium chloride cytochemistry and transmission electron microscopy (TEM). ALP was found in 2 distinct cytoplasmic compartments. One compartment displayed morphology consistent with a subpopulation of secondary granules while a second tubulo-vesicular population appeared similar to the secretory vesicles of human neutrophils. Thin tubular vesicles containing ALP were also identified within the cytoplasm of tammar wallaby eosinophils. Large numbers of ALP-containing vesicles have not been recognised previously in eosinophils and this may represent a novel cytoplasmic compartment. In both cell types, ALP-containing structures showed alteration in morphology following stimulation with N-formyl-Met-Leu-Phe (fMLP) and PMA.

Expressed sequence identification and characterization of the cDNA for interleukin-4 from the mitogen-stimulated lymphoid tissue of a marsupial, Macropus eugenii.

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.

Molecular characterization of interleukin-1Beta in the tammar wallaby (Macropus eugenii).

Interleukin-1beta (IL-1ß) 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-1ß in the tammar wallaby (Macropus eugenii). M. eugenii IL-1ß 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-1ß provides fundamental information necessary to progress the study of functional immune responses in this unique group of mammals.

Molecular characterisation of the CD79a and CD79b subunits of the B cell receptor complex in the gray short-tailed opossum (Monodelphis domestica) and tammar wallaby (Macropus eugenii): Delayed B cell immunocompetence in marsupial neonates.

The B cell receptor (BCR) is a multiprotein complex that is pivotal to antigen recognition and signal transduction in B cells. It consists of an antigen binding component, membrane Ig (mIg), non-covalently associated with the signaling component, a disulphide-linked heterodimer of CD79a and CD79b. In this study, the gene and corresponding cDNA for CD79a and CD79b in the gray short-tailed opossum, as well as the cDNA sequences for CD79a and CD79b in the tammar wallaby, are described. Many of the structural and functional features of CD79a and CD79b were conserved in both marsupials, including the ITAM regulatory motif in the cytoplasmic tails of both subunits. The marsupial CD79 sequences shared a high degree of amino acid identities of 76% (CD79a) and 72% (CD79b) to each other, as well as 60-61% (CD79a) and 58-59% (CD79b) with their eutherian counterparts. RT-PCR analysis of CD79a and CD79b transcripts in the immune tissues of tammar pouch young revealed CD79a transcripts in the bone marrow, cervical thymus and spleen at day 10 postpartum. CD79b transcripts were detected in the bone marrow and cervical thymus at day 10 but were not detected in the spleen until day 21 postpartum. These results suggest that a functional BCR may not be assembled until day 21 postpartum and the tammar neonate may not be capable of mounting an effective adaptive immune response until this time. The molecular information presented here will allow further investigation of the role of the CD79 subunits in marsupial B cell signaling, especially during ontogeny and disease.

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.

Immunocontraception of Eastern Grey kangaroos (Macropus giganteus) with recombinant brushtail possum (Trichosurus vulpecula) ZP3 protein.

This study examined the potential of a recombinant marsupial zona pellucida 3 protein as a contraceptive vaccine for the Eastern Grey kangaroo, a marsupial that is locally overabundant in several regions of eastern Australia. First, a pilot study using porcine zona pellucidae (PZP) demonstrated that ZP proteins, primarily the ZP3 component of PZP, are highly immunogenic in the grey kangaroo and produce a long-lasting humoral response to a single immunisation, as found in other marsupials. Immunisation with 300 microg of a non-glycosylated recombinant brushtail possum ZP3 (recBP-ZP3) protein in complete Freund's adjuvant produced a similar, significant and sustained antibody response, and none of the immunised kangaroos (n=7) produced offspring during the following breeding season compared with four out of the six control animals. An epitope analysis of the B-cell response to recBP-ZP3 using a brushtail possum ZP3 identified numerous B-cell epitope regions clustered around the N- and C-terminal regions of the protein. Two regions of interest for further fertility vaccine development based on their immunogenicity and fertility trials and functional studies in other species were found to be immunogenic. These results suggest that immunocontraception based on targeting the ZP3 protein within the zona pellucida may be an effective strategy for fertility reduction in Eastern Grey kangaroos.

The marsupial CD8 gene locus: molecular cloning and expression analysis of the alpha and beta sequences in the gray short-tailed opossum (Monodelphis domestica) and the tammar wallaby (Macropus eugenii).

In eutherian mammals, CD8 is a key receptor of cytotoxic T cells and plays a pivotal role in the recognition and elimination of infected host cells by cell-mediated cytotoxicity. Here, we report the molecular cloning and expression analysis of CD8alpha and CD8beta cDNAs in two marsupial species, the gray short-tailed opossum and the tammar wallaby. The opossum and tammar CD8 sequences share a high degree of amino acid identity of 63% (CD8alpha) and 57% (CD8beta) to each other as well as 36-45% (CD8alpha) and 38-41% (CD8beta) with their eutherian counterparts. In addition, many of the signature features of eutherian CD8alpha and CD8beta are preserved in both marsupials including the two invariant cysteines that form the intra-chain disulphide bond in the extracellular IgSfV domain and the two hinge region cysteines involved in dimerisation between the two subunits. The p56(lck) binding motif in the cytoplasmic tail of the CD8alpha subunit is also conserved. Interestingly, the opossum CD8alpha and the tammar CD8beta sequences have a truncated cytoplasmic tail. RT-PCR analysis of CD8alpha and CD8beta transcripts in the tissues of the adult opossum and tammar showed broad tissue expression with a high level of expression observed in the lymphoid tissues of both marsupials. Furthermore, RT-PCR analysis of CD8alpha and CD8beta transcripts in the immune tissues of tammar young over the first 120 days of pouch life revealed a pattern of expression analogous to the maturation of the lymphoid tissues. This is the first report confirming the presence of CD8 in the tissues of a marsupial and will provide the tools to further analyse T cell subsets in this unique group of mammals.