The common brushtail possum (Trichosurus vulpecula) is a marsupial indigenous to Australia. In New Zealand, the possum is a major invasive pest, causing severe damage to native ecosystems and acting as a vector of bovine tuberculosis (Clout and Ericksen 2000). Various population control methods are being developed for the possum in New Zealand, including immunocontraception (Cowan 2000). The aim of immunocontraception is to generate an autoimmune response that renders the animal infertile (Cowan 2000). Because the immune response is partly under genetic control, an understanding of immunogenetics in the possum will be critical to the development of immunocontraceptive vaccines.

Major histocompatibility complex (MHC) molecules are important in the vertebrate immune response. The eutherian MHC has been well characterised but there is comparatively little known about the MHC in marsupials. Class II MHC molecules bind and present exogenously derived peptides to T lymphocytes. Three classical class II MHC families have been identified in marsupials: DA (possibly equivalent to the eutherian DR), DB and DC (Belov et al. 2004, 2006). Each family consists of genes that encode one or more of the α and β chains that make up the MHC molecule. The peptide binding region (PBR) of class II molecules is formed from the α 1 and β 1 domains of these chains, encoded by exon 2. We have previously described 19 novel alleles from the β chain of the DA locus (DAB) in the possum (Holland et al. 2008). Here, we report on another 11 novel alleles isolated from possum DAB, making this the most diverse marsupial locus described so far.

This work was approved by the Animal Ethics Committee of Landcare Research, Lincoln. All procedures involving animals were carried out in accordance with the 1987 Animal Protection (Codes of Ethical Conduct) Regulations of New Zealand. Forty-nine animals were captured from a wild population in Lewis Pass (42 22.510S,172 24.009E). Mesenteric lymph nodes were collected from the gut connective tissue of 12 of the animals (Table 1). Leukocytes were extracted from lymph nodes by washing nodes with sterile phosphate buffered saline (PBS), stabbing them with a 25-gauge needle attached to a 3-ml syringe and injecting the node with PBS until the cells spilled out. Cells were then transferred to RNAlater (Qiagen) at −20°C. RNA was extracted from cells using the RNeasy Mini Kit (Qiagen) following the manufacturer’s protocol. Complementary DNA (cDNA) was synthesised with the GeneAmp RNA PCR Kit (Applied Biosystems) following the manufacturer’s protocol using random hexamer primers. Samples of ear tissue were collected from the remaining 37 possums as previously described (Holland et al. 2008).

Table 1 Tissues types sampled and DAB alleles found in individual possums
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Exon 2 of possum DAA, DAB, DBA and DBB loci was amplified with the polymerase chain reaction (PCR) primers pairs DAAE2F/DAAE2R, DABE2inF/DABE2inR, DBAE2inF_2/DBAE2inR_2 and DBBE2inF/DBBE2inR, respectively. PCR conditions, visualisation, sequencing and analysis of sequences were as previously described (Holland et al. 2008).

Where there were apparent multiple alleles (due to the presence of single nucleotide polymorphisms), ambiguous sequences were amplified by PCR and then cloned into a pGEM-T Easy vector (Promega) following the manufacturer’s protocol. Clones containing inserts were sequenced in both directions with the PCR primers as detailed in Holland et al. (2008). Eight to 12 clones were sequenced for each individual PCR.

Phylogenetic tree building was conducted with Bayesian and maximum likelihood analyses. The most appropriate model of molecular evolution was first determined in Modeltest 3.7 (Posada and Crandall 1998). Bayesian analysis was conducted in MrBayes 3.11 (Huelsenbeck and Ronquist 2001) for 107 generations. Maximum likelihood analysis was conducted in PAUP* 4.0b10 (Swofford 2002) for ten replicates.

To examine diversity in the PBR of possum class II MHC loci DAA, DAB, DBA and DBB, we determined nucleotide sequence from exon 2 in 49 animals. We identified 11 novel alleles in the DAB locus, which we have named TrvuDAB*011701 to TrvuDAB*012701, and amplified 16 of the previously identified DAB alleles (Table 1; Lam et al. 2001; Holland et al. 2008). No new alleles were found in other loci. All novel DAB sequences were 235 nucleotides long with no insertion/deletion events (indels) in the alleles. Across the 11 novel sequences, there were 96 polymorphic sites containing 118 variants. Fifty-six of the 78 codons contained variations; ten of these were synonymous and 46 were non-synonymous at the amino acid level. When these sequences were combined with the 21 known possum DAB alleles (Lam et al. 2001; Holland et al. 2008), 140 variants were present in 105 polymorphic sites.

Phylogenetic analysis was conducted on all known possum DAB alleles and other marsupial and eutherian MHC loci alleles. Phylogenies constructed using Bayesian and maximum likelihood methods returned identical topologies; therefore, only the Bayesian tree is shown (Fig. 1). As expected, the eutherian DRB, DPB and DQB loci and the marsupial DAB and DBB loci formed separate clades. All possum DAB alleles were found together in a single clade, which also contained other marsupial DAB sequences.

Fig. 1
figure 1

Phylogenetic tree of MHC class II β chain exon 2 alleles. The chicken (GagaBLB) is used as an outgroup. Sequences are given a label containing a species code and locus name. The scale bar gives the number of expected changes per site. Marginal posterior probabilities greater than 0.50 are given for each clade. NCBI accession numbers for sequences are: Marsupial: Trichosurus vulpecula—TrvuDAB1 (AF312030), TrvuDAB2 (AF312029), TrvuDBB (AY271265), Macropus rufogriseus—MaruDAB1 (M81624), MaruDAB2 (M81626), MaruDBB (M81625), Monodelphis domestica—ModoDAB (AF010497), Macropus eugenii—MaeuDBB1 (AY438039), MaeuDBB2 (AY438038), MaeuDBB3 (AY438041); Eutherian: Homo sapiens—HosaDRB (NM_021983), HosaDPB (NM_002121), HosaDQB (NM_002123), Macaca fascicularis—MafaDRB (DQ381751), Macaca mulatta MamuDPB (AB219104), Canis familiaris: CafaDRB (NM_001014768), Bos Taurus—BotaDQB (AY911331), Sus scrofa—SuscDQB (AB009659) and Avian: Gallus gallus—GagaBLB2 (EF579812)

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Conserved features found in the MHC class II loci of other marsupial and eutherian species were present in many of the novel possum DAB alleles. No structural mutations that could disrupt function, such as stop codons or frame-shift mutations, were found in the novel alleles (Fig. 2). All alleles contained conserved paired cysteines (C15, C79) and a potential glycosylation site (NGT, positions 19 to 21), both features of classical class II molecules (Mazerolles et al. 1988; Brogdon et al. 1998; Belov et al. 2004). The potential CD4 interaction site at positions 39 to 42, RFDS (Mazerolles et al. 1988; Brogdon et al. 1998; Belov et al. 2004), was fully conserved in four of the new alleles. The other seven novel DAB alleles featured a single amino acid difference in this motif. The phenylanine at position 40 was replace with a leucine in TrvuDAB*011701 and with a tyrosine in TrvuDAB*012501. The aspartic acid at position 41 was an aspargine in TrvuDAB*011801 and TrvuDAB*012401 and a tyrosine in TrvuDAB*012001. The serine at position 42 was changed to a glycine in TrvuDAB*012301 and TrvuDAB*012601. Many of these changes are non-conservative; however, the marsupial CD4 has unique features (Duncan et al. 2007), and the impact of this variation on the function of these MHC molecules is unknown. It has been suggested that in eutherians, a tryptophan (W61) and an aspargine (N82) bond with the main chain of bound peptides (Brown et al. 1993). In the possum, both positions vary; position 61 can be tryptophan, tyrosine, leucine or glycine and position 82 can be aspargine or tryptophan. Variation at position 61 has been previously reported in marsupial species, and it is unknown what affect differences in the amino acids at these positions may have on peptide binding (Lam et al. 2001; Siddle et al. 2007).

Fig. 2
figure 2

Alignment of deduced amino acid class II DAB sequences of the possum and human DRB. Alpha helix and beta pleated sheet structures determined for TrvuDAB*010101 are shown above the sequences with a helix symbol and arrow, respectively. Alpha helix and beta-pleated sheet structures were determined by investigating secondary structure models. A potential glycosylation site (NGT) and potential CD4 interaction site (RFDS; Mazerolles et al. 1988; Brogdon et al. 1998; Belov et al. 2004) are boxed. Conserved cysteine residues are indicated with a C and peptide binding residues (Brown et al. 1993) are indicated with a B below the sequence. Dots indicate sequence agreement with TrvuDAB2; dashes indicate gaps in the sequence introduced to optimize the alignment

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More variations were observed in the DAB locus than in other possum MHC class II loci investigated. In the loci DAA, DBA and DBB, no novel sequences were identified (data not shown). In total, five alleles have been isolated in the possum from DAA, 12 from DBA, five from DBB and 32 from DAB (Belov et al. 2004; Holland et al. 2008). Comparing the level of polymorphism in the PBRs of the α and β chain loci shows the DAB locus had the highest number of substitutions (Fig. 3). In the α chain loci, DAA had a single polymorphic amino acid position out of the 82 available (0.012) and in DBA 26 of the 70 available positions (0.371) were polymorphic. In the β chain loci, 49 of the 78 available positions (0.628) were polymorphic in DAB, compared to 23 of the available 90 positions (0.256) in DBB. More than two substitutions were found in only one position of the DBB locus but in 18 positions of the DAB locus. Polymorphic positions found in possum DAB agree well with those identified in human DRB (Brown et al. 1993), suggesting a similar arrangement of polymorphic pockets occurs in the possum.

Fig. 3
figure 3

Substitutions in polymorphic sites of the PBR in the class II MHC loci of the possum. a Alpha chain loci DAA and DBA; b beta chain loci DAB and DBB. Amino acid position is numbered from the beginning of the β 1 domain (Brown et al. 1993)

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Up to six DAB alleles were found in individual possums (Table 1). Some species are known to possess multiple copies of MHC genes, an example being the nine DRB loci known from humans (Campbell and Trowsdale 1993; Marsh et al. 2000). The presence of greater than two alleles in an individual suggests that possums have multiple DAB genes. We have previously found that multiple genes are likely to be present in possum DAB, DBA and DBB loci (Holland et al. 2008).

We have found a large amount of diversity in possum MHC genes. The MHC is known to be highly variable (Marsh et al. 2000). As a relatively recently introduced population, possums in New Zealand might be expected to have limited genetic variation. However, at least 35 importations of possums to New Zealand were made during the 1800s (McDowall 1994). Importations were from a range of locations in eastern Australia and Tasmania and that there was much human assisted mixing of populations in the 50 years after introductions (Clout and Ericksen 2000). This large number of independent importations means considerable genetic variation may have been present at the outset of possum colonisation of New Zealand, with the variability we have identified reflecting pre-importation variation. Supporting this contention, high variability in possum microsatellite markers has also been found in New Zealand (Taylor et al. 2004), indicating wide genetic diversity in New Zealand possums.

The 11 novel possum DAB alleles we identified with the 21 previously isolated possum DAB alleles (Lam et al. 2001; Holland et al. 2008) raises the total alleles now reported from this locus to 32. Therefore, DAB is the most diverse marsupial locus described so far. The large number of substitutions and high ratio on non-synonymous changes found in these alleles suggests that this locus is under positive selection for diversity. Such high levels of diversity indicate that DAB is an important MHC locus in the possum.

The wide diversity in the MHC of the New Zealand possum population may need to be taken into account in future work aimed at developing immunocontraceptive vaccines for use in possum population control, and means care will need to be taken to ensure any contraceptive vaccines are effective for a wide range of possum MHC haplotypes. We are currently investigating how individual differences in response to experimental immunocontraceptive vaccines (Cui and Duckworth 2005; Duckworth et al. 2007) are impacted by genetic diversity in DAB and other MHC loci. The nucleotide sequences reported in this article have been submitted to the GenBank nucleotide sequence database and have been assigned accession numbers ranging from EU849585 to EU849595.