, Volume 65, Issue 1, pp 37–46 | Cite as

Characterisation of four major histocompatibility complex class II genes of the koala (Phascolarctos cinereus)

  • Quintin Lau
  • Sarah E. Jobbins
  • Katherine Belov
  • Damien P. HigginsEmail author
Original Paper


Major histocompatibility complex (MHC) class II molecules have an integral role in the adaptive immune response, as they bind and present antigenic peptides to T helper lymphocytes. In this study of koalas, species-specific primers were designed to amplify exon 2 of the MHC class II DA and DB genes, which contain much of the peptide-binding regions of the α and β chains. A total of two DA α1 domain variants and eight DA β1 (DAB), three DB α1 and five DB β1 variants were amplified from 20 koalas from two free-living populations from South East Queensland and the Port Macquarie region in northern New South Wales. We detected greater variation in the β1 than in the α1 domains as well as evidence of positive selection in DAB. The present study provides a springboard to future investigation of the role of MHC in disease susceptibility in koalas.


Koala Phascolarctos cinereus MHC class II Marsupial Diversity 



This research was funded by the Hermon Slade Foundation. All procedures were approved by the University of Sydney Animal Ethics Committee (AEC N00/4-2005/3/4088, AEC N00/5-2009/1/4829).We acknowledge Weerachai Jaratlerdsiri and Simon Ho for their help with analyses, Jaime Gongora for guidance on the manuscript and Joanna Griffith and all staff at the koala hospitals for sample collection.

Supplementary material

251_2012_658_MOESM1_ESM.pdf (792 kb)
ESM 1 (PDF 792kb)


  1. Balakrishnan K, Adams LE (1995) The role of the lymphocyte in an immune response. Immunol Investig 24(1–2):233–244. doi: 10.3109/08820139509062775 CrossRefGoogle Scholar
  2. Belov K, Lam MK-P, Colgan DJ (2004) Marsupial MHC class II β genes are not orthologous to the eutherian β gene families. J Hered 95(4):338–345. doi: 10.1093/jhered/esh049 PubMedCrossRefGoogle Scholar
  3. Belov K, Deakin JE, Papenfuss AT, Baker ML, Melman SD, Siddle HV, Gouin N, Goode DL, Sargeant TJ, Robinson MD, Wakefield MJ, Mahony S, Cross JG, Benos PV, Samollow PB, Speed TP, Graves JA, Miller RD (2006) Reconstructing an ancestral mammalian immune supercomplex from a marsupial major histocompatibility complex. PLoS Biol 4(3):e46. doi: 10.1371/journal.pbio.0040046 PubMedCrossRefGoogle Scholar
  4. Brown JH, Jardetzky TS, Gorga JC, Stern LJ, Urban RG, Strominger JL, Wiley DC (1993) Three-dimensional structure of the human class II histocompatibility antigen HLA-DR1. Nature 364(6432):33–39. doi: 10.1038/364033a0 PubMedCrossRefGoogle Scholar
  5. Browning TL (2009) Inbreeding, the MHC and infectious disease susceptibility in the tammar wallaby, Macropus eugenii. Macquarie University, SydneyGoogle Scholar
  6. Browning TL, Belov K, Miller RD, Eldridge MDB (2004) Molecular cloning and characterization of the polymorphic MHC class II DBB from the tammar wallaby (Macropus eugenii). Immunogenetics 55:791–795. doi: 10.1007/s00251-004-0644-7 PubMedCrossRefGoogle Scholar
  7. Canfield PJ, Hemsley S, Connolly J (1996) Histological and immunohistological study of the developing and involuting superficial cervical thymus in the koala (Phascolarctos cinereus). J Anat 189:159–169PubMedGoogle Scholar
  8. Cheng Y, Siddle HV, Beck S, Eldridge MD, Belov K (2009) High levels of genetic variation at MHC class II DBB loci in the tammar wallaby (Macropus eugenii). Immunogenetics 61(2):111–118. doi: 10.1007/s00251-008-0347-6 PubMedCrossRefGoogle Scholar
  9. Dique DS, Thompson J, Preece HJ, Penfold GC, De Villiers DL, Leslie RS (2003) Koala mortality on roads in south-east Queensland: the koala speed-zone trial. Wildl Res 30(4):419–426. doi: 10.1071/WR02029 CrossRefGoogle Scholar
  10. Griffith JE, Dhand NK, Krockenberger MB, Higgins DP (2013) A retrospective study of admission trends of koalas to a rehabilitation facility over thirty years. J Wildl Dis 49(1):doi: 10.7589/2012-05-135 Google Scholar
  11. Higgins DP, Hemsley S, Canfield PJ (2005) Association of uterine and salpingeal fibrosis with chlamydial hsp60 and hsp10 antigen-specific antibodies in Chlamydia-infected koalas. Clin Diagn Lab Immunol 12(5):632–639. doi: 10.1128/CDLI.12.5.632-639.2005 PubMedGoogle Scholar
  12. Holland OJ, Cowan PE, Gleeson DM, Chamley LW (2008) Novel alleles in classical major histocompatibility complex class II loci of the brushtail possum (Trichosurus vulpecula). Immunogenetics 60(8):449–460. doi: 10.1007/s00251-008-0300-8 PubMedCrossRefGoogle Scholar
  13. Houlden BA, England PR, Taylor AC, Greville WD, Sherwin WB (1996) Low genetic variability of the koala Phascolarctos cinereus in south-eastern Australia following a severe population bottleneck. Mol Ecol 5(2):269–281PubMedGoogle Scholar
  14. Huelsenbeck JP, Ronquist F (2001) MRBAYES: Bayesian inference of phylogenetic trees. Bioinformatics 17(8):754–755PubMedCrossRefGoogle Scholar
  15. Hughes AL, Friedman R (2008) Codon-based tests of positive selection, branch lengths, and the evolution of mammalian immune system genes. Immunogenetics 60(9):495–506. doi: 10.1007/s00251-008-0304-4 PubMedCrossRefGoogle Scholar
  16. Jackson M, White N, Giffard P, Timms P (1999) Epizootiology of Chlamydia infections in two free-range koala populations. Vet Microbiol 65(4):255–264. doi: S0378113598003022 PubMedCrossRefGoogle Scholar
  17. Jobbins SE, Sanderson CE, Griffith JE, Krockenberger MB, Belov K, Higgins DP (2012) Diversity of MHC class II DAB1 in the koala (Phascolarctos cinereus). Aust J Zool 60(1):1–9. doi: 10.1071/ZO12013 CrossRefGoogle Scholar
  18. Kalish RS (1995) Antigen processing: the gateway to the immune response. J Am Acad Dermatol 32(4):640–652PubMedCrossRefGoogle Scholar
  19. Keane T, Creevey C, Pentony M, Naughton T, Mclnerney J (2006) Assessment of methods for amino acid matrix selection and their use on empirical data shows that ad hoc assumptions for choice of matrix are not justified. BMC Evol Biol 6(1):29. doi: 10.1186/1471-2148-6-29 PubMedCrossRefGoogle Scholar
  20. Kennedy LJ, Ryvar R, Gaskell RM, Addie DD, Willoughby K, Carter SD, Thomson W, Ollier WE, Radford AD (2002) Sequence analysis of MHC DRB alleles in domestic cats from the United Kingdom. Immunogenetics 54(5):348–352. doi: 10.1007/s00251-002-0465-5 PubMedCrossRefGoogle Scholar
  21. Klein J, Bontrop RE, Dawkins RL, Erlich HA, Gyllensten UB, Heise ER, Jones PP, Parham P, Wakeland EK, Watkins DI (1990) Nomenclature for the major histocompatibility complexes of different species: a proposal. Immunogenetics 31(4):217–219PubMedCrossRefGoogle Scholar
  22. Lam MK, Belov K, Harrison GA, Cooper DW (2001) Cloning of the MHC class II DRB cDNA from the brushtail possum (Trichosurus vulpecula). Immunol Lett 76(1):31–36. doi: S0165-2478(00)00314-X PubMedCrossRefGoogle Scholar
  23. Lau Q, Canfield PJ, Higgins DP (2012) Expression and in vitro upregulation of MHCII in koala lymphocytes. Vet Immunol Immunopathol 147(1–2):35–43. doi: 10.1016/j.vetimm.2012.04.010 PubMedCrossRefGoogle Scholar
  24. Lee A, Martin R (1988) The koala a natural history. New South Wales University Press, KensingtonGoogle Scholar
  25. Lillie M, Woodward RE, Sanderson CE, Eldridge MDB, Belov K (2012) Diversity at the major histocompatibility complex class II in the platypus, Ornithorhynchus anatinus. J Hered 103(4):467–478. doi: 10.1093/jhered/ess012 PubMedCrossRefGoogle Scholar
  26. Lunney D, Gresser S, O’Neill L, Matthews A, Rhodes JR (2007) The impact of fire and dogs on koalas at Port Stephens, New South Wales, using population viability analysis. Pac Conserv Biol 13(3):189–201Google Scholar
  27. Marsh J, Kollipara A, Timms P, Polkinghorne A (2011) Novel molecular markers of Chlamydia pecorum genetic diversity in the koala (Phascolarctos cinereus). BMC Microbiol 11(1):77. doi: 10.1186/1471-2180-11-77 PubMedCrossRefGoogle Scholar
  28. Mazerolles F, Durandy A, Piatier-Tonneau D, Charron D, Montagnier L, Auffray C, Fischer A (1988) Immunosuppressive properties of synthetic peptides derived from CD4 and HLA-DR antigens. Cell 55(3):497–504PubMedCrossRefGoogle Scholar
  29. Melzer A, Carrick F, Menkhorst P, Lunney D, John BS (2000) Overview, critical assessment, and conservation implications of koala distribution and abundance. Conserv Biol 14(3):619–628. doi: 10.1046/j.1523-1739.2000.99383.x CrossRefGoogle Scholar
  30. Meyer-Lucht Y, Otten C, Puttker T, Sommer S (2008) Selection, diversity and evolutionary patterns of the MHC class II DAB in free-ranging Neotropical marsupials. BMC Genet 9:39. doi: 10.1186/1471-2156-9-39 PubMedCrossRefGoogle Scholar
  31. Meyer-Lucht Y, Otten C, Püttker T, Pardini R, Metzger J, Sommer S (2010) Variety matters: adaptive genetic diversity and parasite load in two mouse opossums from the Brazilian Atlantic forest. Conserv Genet 11(5):2001–2013. doi: 10.1007/s10592-010-0093-9 CrossRefGoogle Scholar
  32. Miller HC, Andrews-Cookson M, Daugherty CH (2007) Two patterns of variation among MHC class I loci in Tuatara (Sphenodon punctatus). J Hered 98(7):666–677. doi: 10.1093/jhered/esm095 PubMedCrossRefGoogle Scholar
  33. Mitchell P (1990) Social behaviour and communication of koalas. In: Lee AK, Handasyde KA, Sanson GD (eds) Biology of the koala. Surrey Beatty, Chipping Norton, (Australia), pp 151-170Google Scholar
  34. Morrison RP, Feilzer K, Tumas DB (1995) Gene knockout mice establish a primary protective role for major histocompatibility complex class II-restricted responses in Chlamydia trachomatis genital tract infection. Infect Immun 63(12):4661–4668PubMedGoogle Scholar
  35. O’Brien SJ, Evermann JF (1988) Interactive influence of infectious disease and genetic diversity in natural populations. Trends Ecol Evol 3(10):254–259PubMedCrossRefGoogle Scholar
  36. O’HUigin C, Sultmann H, Tichy H, Murray BW (1998) Isolation of MHC class II DMA and DMB cDNA sequences in a marsupial: the gray short-tailed opossum (Monodelphis domestica). J Mol Evol 47(5):578–585PubMedCrossRefGoogle Scholar
  37. Obendorf DL, Handasyde KA (1990) Pathology of chlamydial infection in the reproductive tract of the female koala. In: Lee AJ, Handasyde KA, Sanson GD (eds) Biology of the koala. Surrey and Beatty and Sons, Sydney, pp 255–259Google Scholar
  38. Ohta T (1998) On the pattern of polymorphisms at major histocompatibility complex loci. J Mol Evol 46(6):633–638PubMedCrossRefGoogle Scholar
  39. Orita M, Iwahana H, Kanazawa H, Hayashi K, Sekiya T (1989) Detection of polymorphisms of human DNA by gel electrophoresis as single-strand conformation polymorphisms. Proc Natl Acad Sci U S A 86(8):2766–2770PubMedCrossRefGoogle Scholar
  40. Rambaut A (2009) FigTree 1.3.1 ednGoogle Scholar
  41. Schneider S, Vincek V, Tichy H, Figueroa F, Klein J (1991) MHC class II genes of a marsupial, the red-necked wallaby (Macropus rufogriseus): identification of new gene families. Mol Biol Evol 8(6):753–766PubMedGoogle Scholar
  42. Siddle HV, Sanderson C, Belov K (2007) Characterization of major histocompatibility complex class I and class II genes from the Tasmanian devil (Sarcophilus harrisii). Immunogenetics 59:753–760. doi: 10.1007/s00251-007-0238-2 PubMedCrossRefGoogle Scholar
  43. Siddle HV, Deakin JE, Coggill P, Whilming L, Harrow J, Kaufman J, Beck S, Belov K (2011) The tammar wallaby major histocompatibility complex shows evidence of past genomic instability. BMC Genomics 12:421. doi: 10.1186/1471-2164-12-421 PubMedCrossRefGoogle Scholar
  44. Slade RW, Mayer WE (1995) The expressed class II alpha-chain genes of the marsupial major histocompatibility complex belong to eutherian mammal gene families. Mol Biol Evol 12(3):441–450PubMedGoogle Scholar
  45. Sommer S (2005) The importance of immune gene variability (MHC) in evolutionary ecology and conservation. Front Zool 2(16):doi: 10.1186/1742-9994-2-16 Google Scholar
  46. Stamatakis A (2006) RAxML-VI-HPC: maximum likelihood-based phylogenetic analyses with thousands of taxa and mixed models. Bioinformatics 22(21):2688–2690. doi: 10.1093/bioinformatics/btl446 PubMedCrossRefGoogle Scholar
  47. Stephens RS (2003) The cellular paradigm of chlamydial pathogenesis. Trends Microbiol 11(1):44–51. doi: 10.1016/S0966-842X(02)00011-2 PubMedCrossRefGoogle Scholar
  48. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28(10):2731–2739. doi: 10.1093/molbev/msr121 PubMedCrossRefGoogle Scholar
  49. Zhang J, Nielsen R, Yang Z (2005) Evaluation of an improved branch-site likelihood method for detecting positive selection at the molecular level. Mol Biol Evol 22(12):2472–2479. doi: 10.1093/molbev/msi237 PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2012

Authors and Affiliations

  • Quintin Lau
    • 1
  • Sarah E. Jobbins
    • 1
  • Katherine Belov
    • 1
  • Damien P. Higgins
    • 1
    Email author
  1. 1.Faculty of Veterinary ScienceThe University of SydneyNSWAustralia

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