Australian Journal of Zoology Australian Journal of Zoology Society
Evolutionary, molecular and comparative zoology
RESEARCH ARTICLE

The koala immunological toolkit: sequence identification and comparison of key markers of the koala (Phascolarctos cinereus) immune response

Katrina Morris A , Peter J. Prentis B , Denis O’Meally A , Ana Pavasovic C , Alyce Taylor Brown D , Peter Timms D E , Katherine Belov A and Adam Polkinghorne D E F

A Faculty of Veterinary Science, University of Sydney, RMC Gunn, B19, Sydney, NSW 2006, Australia.

B School of Earth, Environmental and Biological Sciences, Queensland University of Technology, 2 George Street, Brisbane, Qld 4001, Australia.

C School of Biomedical Sciences, Queensland University of Technology, 2 George Street, Brisbane, Qld 4001, Australia.

D Institute of Health and Biomedical Innovation, Queensland University of Technology, 60 Musk Avenue, Kelvin Grove, Qld 4059, Australia.

E Faculty of Science, Health, Education and Engineering, University of the Sunshine Coast, 90 Sippy Downs Drive, Sippy Downs, Qld 4556, Australia.

F Corresponding author. Email: a.polkinghorne@qut.edu.au

Australian Journal of Zoology 62(3) 195-199 http://dx.doi.org/10.1071/ZO13105
Submitted: 6 December 2013  Accepted: 30 March 2014   Published: 28 April 2014

Abstract

The koala (Phascolarctos cinereus) is an Australian marsupial that continues to experience significant population declines. Infectious diseases caused by pathogens such as Chlamydia are proposed to have a major role. Very few species-specific immunological reagents are available, severely hindering our ability to respond to the threat of infectious diseases in the koala. In this study, we utilise data from the sequencing of the koala transcriptome to identify key immunological markers of the koala adaptive immune response and cytokines known to be important in the host response to chlamydial infection in other species. This report describes the identification and preliminary sequence analysis of (1) T lymphocyte glycoprotein markers (CD4, CD8); (2) IL-4, a marker for the Th2 response; (3) cytokines such as IL-6, IL-12 and IL-1β, that have been shown to have a role in chlamydial clearance and pathology in other hosts; and (4) the sequences for the koala immunoglobulins, IgA, IgG, IgE and IgM. These sequences will enable the development of a range of immunological reagents for understanding the koala’s innate and adaptive immune responses, while also providing a resource that will enable continued investigations into the origin and evolution of the marsupial immune system.

Graphical Abstract Image


References

Cisternas, P. A., and Armati, P. J. (2000). Immune system cell markers in the northern brown bandicoot, Isoodon macrourus. Developmental and Comparative Immunology 24, 771–782.
Immune system cell markers in the northern brown bandicoot, Isoodon macrourus.CrossRef | 1:CAS:528:DC%2BD3cXkvFSltLo%3D&md5=1e6e2ddc7aa0698c6f43f4fca9c409baCAS | 10906390PubMed | open url image1

Cunningham, K., Stansfield, S. H., Patel, P., Menon, S., Kienzle, V., Allan, J. A., and Huston, W. M. (2013). The IL-6 response to Chlamydia from primary reproductive epithelial cells is highly variable and may be involved in differential susceptibility to the immunopathological consequences of chlamydial infection. BMC Immunology 14, 50.
The IL-6 response to Chlamydia from primary reproductive epithelial cells is highly variable and may be involved in differential susceptibility to the immunopathological consequences of chlamydial infection.CrossRef | 24238294PubMed | open url image1

Duncan, L. G., Nair, S. V., and Deane, E. M. (2007). Molecular characterisation and expression of CD4 in two distantly related marsupials: the gray short-tailed opossum (Monodelphis domestica) and tammar wallaby (Macropus eugenii). Molecular Immunology 44, 3641–3652.
Molecular characterisation and expression of CD4 in two distantly related marsupials: the gray short-tailed opossum (Monodelphis domestica) and tammar wallaby (Macropus eugenii).CrossRef | 1:CAS:528:DC%2BD2sXmtVSks7s%3D&md5=0344fb7251d5a935d77d55df0d9148a1CAS | 17521733PubMed | open url image1

Duncan, L. G., Nair, S. V., and Deane, E. M. (2009). 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). Veterinary Immunology and Immunopathology 129, 14–27.
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).CrossRef | 1:CAS:528:DC%2BD1MXjsFWlsro%3D&md5=2fafbbf8dcc19692011f455d22640300CAS | 19135263PubMed | open url image1

Gordon, G., Hrdina, F., and Patterson, R. (2006). Decline in the distribution of the koala Phascolarctos cinereus in Queensland. Australian Zoologist 33, 345–358.
Decline in the distribution of the koala Phascolarctos cinereus in Queensland.CrossRef | open url image1

Grabherr, M. G., Haas, B. J., Yassour, M., Levin, J. Z., Thompson, D. A., Amit, I., Adiconis, X., Fan, L., Raychowdury, R., Zeng, Q., Chen, Z., Mauceli, E., Hacohen, N., Gnirke, A., Rhind, N., di Palma, F., Birren, B. W., Nusbaum, C., Lindblad-Toh, K., Friedman, N., and Regev, A. (2011). Full-length transcriptome assembly from RNA-Seq data without a reference genome. Nature Biotechnology 29, 644–652.
Full-length transcriptome assembly from RNA-Seq data without a reference genome.CrossRef | 1:CAS:528:DC%2BC3MXmtV2hsbc%3D&md5=b7d133aa2366041cec36e44ea80dd042CAS | 21572440PubMed | open url image1

Hall, T. (1999). Bioedit: a user friendly biological sequence alignment editor and analysis program for windows 95/98/NT. Nucleic Acids Symposium Series 41, 95–98.
| 1:CAS:528:DC%2BD3cXhtVyjs7Y%3D&md5=1bc8594ada2830f08a4bc74ad945df91CAS | open url image1

Howson, L. J., Morris, K. M., Kobayashi, T., Tovar, C., Kreiss, A., Papenfuss, A. T., Corcoran, L., Belov, K., and Woods, G. M. (2013). Identification of dendritic cells, B cell and T cell subsets in Tasmanian devil lymphoid tissue; evidence for poor immune cell infiltration into devil facial tumors. The Anatomical Record , .
Identification of dendritic cells, B cell and T cell subsets in Tasmanian devil lymphoid tissue; evidence for poor immune cell infiltration into devil facial tumors.CrossRef | open url image1

Khader, S. A., and Gopal, R. (2010). IL-17 in protective immunity to intracellular pathogens. Virulence 1, 423–427.
IL-17 in protective immunity to intracellular pathogens.CrossRef | 21178483PubMed | open url image1

Kollipara, A., George, C., Hanger, J., Loader, J., Polkinghorne, A., Beagley, K., and Timms, P (2012). Vaccination of healthy and diseased koalas (Phascolarctos cinereus) with a Chlamydia pecorum multi-subunit vaccine: evaluation of immunity and pathology. Vaccine 30, 1875–1885.
Vaccination of healthy and diseased koalas (Phascolarctos cinereus) with a Chlamydia pecorum multi-subunit vaccine: evaluation of immunity and pathology.CrossRef | 1:CAS:528:DC%2BC38XivVygt78%3D&md5=a0cb08a4d09f206bc62a307c2d4efaa3CAS | 22230583PubMed | open url image1

Lau, Q., Jobbins, S. E., Belov, K., and Higgins, D. P. (2013). Characterisation of four major histocompatibility complex class II genes of the koala (Phascolarctos cinereus). Immunogenetics 65, 37–46.
Characterisation of four major histocompatibility complex class II genes of the koala (Phascolarctos cinereus).CrossRef | 1:CAS:528:DC%2BC3sXlt1KqsQ%3D%3D&md5=7ebb72990275f7ed301fdf4b41852d4cCAS | 23089959PubMed | open url image1

Lu, H, and Zhong, G (1999). Interleukin-12 production is required for chlamydial antigen-pulsed dendritic cells to induce protection against live Chlamydia trachomatis infection. Infection and Immunity 67, 1763–1769.
| 1:CAS:528:DyaK1MXitFCrsL8%3D&md5=ba88b6b841e1a4bbae4b3ee067e619f0CAS | 10085016PubMed | open url image1

Mathew, M., Beagley, K. W., Timms, P., and Polkinghorne, A. (2013a). Preliminary characterisation of tumor necrosis factor alpha and interleukin-10 responses to Chlamydia pecorum infection in the koala (Phascolarctos cinereus). PLoS ONE 8, e59958.
Preliminary characterisation of tumor necrosis factor alpha and interleukin-10 responses to Chlamydia pecorum infection in the koala (Phascolarctos cinereus).CrossRef | 1:CAS:528:DC%2BC3sXltFykurc%3D&md5=3aee68535e86507947f046b6477f4b47CAS | 23527290PubMed | open url image1

Mathew, M., Pavasovic, A., Prentis, P. J., Beagley, K. W., Timms, P., and Polkinghorne, A. (2013b). Molecular characterisation and expression analysis of interferon gamma in response to natural Chlamydia infection in the koala, Phascolarctos cinereus. Gene 527, 570–577.
Molecular characterisation and expression analysis of interferon gamma in response to natural Chlamydia infection in the koala, Phascolarctos cinereus.CrossRef | 1:CAS:528:DC%2BC3sXhtFamu7fO&md5=4710cbea9ce61c39fd7c13a04f19bbc4CAS | 23792018PubMed | open url image1

Melzer, A., Carrick, F., Menkhorst, P., Lunney, D., and John, B. S. (2000). Overview, critical assessment and conservation implications of koala distribution and abundance. Conservation Biology 14, 619–628.
Overview, critical assessment and conservation implications of koala distribution and abundance.CrossRef | open url image1

Murchison, E. P., Schulz-Trieglaff, O. B., Ning, Z., Alexandrov, L. B., Bauer, M. J., Fu, B., Hims, M., Ding, Z., Ivakhno, S., Stewart, C., Ng, B. L., Wong, W., Aken, B., White, S., Alsop, A., Becq, J., Bignell, G. R., Cheetham, R. K., Cheng, W., Connor, T. R., Cox, A. J., Feng, Z. P., Gu, Y., Grocock, R. J., Harris, S. R., Khrebtukova, I., Kingsbury, Z., Kowarsky, M., Kreiss, A., Luo, S., Marshall, J., McBride, D. J., Murray, L., Pearse, A. M., Raine, K., Rasolonjatovo, I., Shaw, R., Tedder, P., Tregidgo, C., Vilella, A. J., Wedge, D. C., Woods, G. M., Gormley, N., Humphray, S., Schroth, G., Smith, G., Hall, K., Searle, S. M., Carter, N. P., Papenfuss, A. T., Futreal, P. A., Campbell, P. J., Yang, F., Bentley, D. R., Evers, D. J., and Stratton, M. R. (2012). Genome sequencing and analysis of the Tasmanian devil and its transmission cancer. Cell 148, 780–791.
Genome sequencing and analysis of the Tasmanian devil and its transmission cancer.CrossRef | 1:CAS:528:DC%2BC38XjtV2qsLs%3D&md5=065637dcd3911a09ba5ca1604bf83027CAS | 22341448PubMed | open url image1

Nelms, K., Keegan, A. D., Zamorano, J., Ryan, J. J., and Paul, W. E. (1999). The IL-4 receptor: signaling mechanisms and biologic functions. Annual Review of Immunology 17, 701–738.
The IL-4 receptor: signaling mechanisms and biologic functions.CrossRef | 1:CAS:528:DyaK1MXjtVWmtLs%3D&md5=5fff71d88e731869715f7cc1700c261bCAS | 10358772PubMed | open url image1

Old, J. M., and Deane, E. M. (2002). Immunohistochemistry of the lymphoid tissues of the tammar wallaby, Macropus eugenii. Journal of Anatomy 201, 257–266.
Immunohistochemistry of the lymphoid tissues of the tammar wallaby, Macropus eugenii.CrossRef | 12363276PubMed | open url image1

Papenfuss, A. T., Baker, M. L., Feng, Z.-P., Tachedjian, M., Crameri, G., Cowled, C., Ng, J., Janardhana, V., Field, H. E., and Wang, L.-F. (2012). The immune gene repertoire of an important viral reservoir, the Australian black flying fox. BMC Genomics 13, 261.
The immune gene repertoire of an important viral reservoir, the Australian black flying fox.CrossRef | 1:CAS:528:DC%2BC38Xhslamu77O&md5=6dc73cc631675d6c11659874341f357cCAS | 22716473PubMed | open url image1

Petersen, T. N., Brunak, S., von Heijne, G., and Nielsen, H. (2011). SignalP 4.0: discriminating signal peptides from transmembrane regions. Nature Methods 8, 785–786.
SignalP 4.0: discriminating signal peptides from transmembrane regions.CrossRef | 1:CAS:528:DC%2BC3MXht1CrtrbL&md5=ec3386b5603129408ec693e86c41eb32CAS | 21959131PubMed | open url image1

Polkinghorne, A., Hanger, J., and Timms, P. (2013). Recent advances in understanding the biology, epidemiology and control of chlamydial infections in the koala. Veterinary Microbiology 165, 214–223.
Recent advances in understanding the biology, epidemiology and control of chlamydial infections in the koala.CrossRef | 23523170PubMed | open url image1

Renfree, M.B., Papenfuss, A.T., Deakin, J.E., Lindsay, J., Heider, T., Belov, K., Rens, W., Waters, P.D., Pharo, E.A., Shaw, G., Wong, E.S., Lefèvre, C.M., Nicholas, K.R., Kuroki, Y., Wakefield, M.J., Zenger, K.R., Wang, C., Ferguson-Smith, M., Nicholas, F.W., Hickford, D., Yu, H., Short, K.R., Siddle, H.V., Frankenberg, S.R., Chew, K.Y., Menzies, B.R., Stringer, J.M., Suzuki, S., Hore, T.A., Delbridge, M.L., Patel, H.R., Mohammadi, A., Schneider, N.Y., Hu, Y., O’Hara, W., Al Nadaf, S., Wu, C., Feng, Z.P., Cocks, B.G., Wang, J., Flicek, P., Searle, S.M., Fairley, S., Beal, K., Herrero, J., Carone, D.M., Suzuki, Y., Sugano, S., Toyoda, A., Sakaki, Y., Kondo, S., Nishida, Y., Tatsumoto, S., Mandiou, I., Hsu, A., McColl, K.A., Lansdell, B., Weinstock, G., Kuczek, E, McGrath, A, Wilson, P, Men, A, Hazar-Rethinam, M, Hall, A, Davis, J, Wood, D, Williams, S, Sundaravandanam, Y, Muzny, D.M., Jhangiani, S.N., Lewis, L.R., Morgan, M.B., Okwuonu, G.O., Ruiz, S.J., Santibanez, J, Nazareth, L, Cree, A, Fowler, G, Kovar, C.L., Dinh, H.H., Joshi, V, Jing, C, Lara, F, Thornton, R, Chen, L, Deng, J, Liu, Y, Shen, J.Y., Song, X.Z., Edson, J, Troon, C, Thomas, D, Stephens, A, Yapa, L, Levchenko, T, Gibbs, R.A., Cooper, D.W., Speed, T.P., Fujiyama, A, Graves, J.A., O’Neill, R.J., Pask, A.J., Forrest, S.M., and Worley, K.C. (2011). Genome sequence of an Australian kangaroo, Macropus eugenii, provides insight into the evolution of mammalian reproduction and development. Genome Biology 12, R81.
Genome sequence of an Australian kangaroo, Macropus eugenii, provides insight into the evolution of mammalian reproduction and development.CrossRef | 21854559PubMed | open url image1

Rhodes, J. R., Ng, C. F., de Villiers, D. L., Preece, H. J., McAlpine, C. A., and Possingham, H. P. (2011). Using integrated population modelling to quantify the implications of multiple threatening processes for a rapidly declining population. Biological Conservation 144, 1081–1088.
Using integrated population modelling to quantify the implications of multiple threatening processes for a rapidly declining population.CrossRef | open url image1

Romano, T. A., Ridgway, S. H., Felten, D. L., and Quaranta, V. (1999). Molecular cloning and characterization of CD4 in an aquatic mammal, the white whale Delphinapterus leucas. Immunogenetics 49, 376–383.
Molecular cloning and characterization of CD4 in an aquatic mammal, the white whale Delphinapterus leucas.CrossRef | 1:CAS:528:DyaK1MXitVKmsbk%3D&md5=ecbd6f182ed74575db243c19f3c27c34CAS | 10199913PubMed | open url image1

Shimada, K., Crother, T. R., Karlin, J., Chen, S., Chiba, N., Ramanujan, V. K., Vergnes, L., and Ojcius, D. M. (2011). Caspase-1 dependent IL-1β secretion is critical for host defence in a mouse model of Chlamydia pneumoniae lung infection. PLoS ONE 6, e21477.
Caspase-1 dependent IL-1β secretion is critical for host defence in a mouse model of Chlamydia pneumoniae lung infection.CrossRef | 1:CAS:528:DC%2BC3MXotF2ns70%3D&md5=7f365a08ec25b3e831c44ebf25a2c1d6CAS | 21731762PubMed | open url image1

Sun, Y., Wei, Z., Li, N., and Zhao, Y. (2013). A comparative overview of immunoglobulin genes and the generation of their diversity in tetrapods. Developmental and Comparative Immunology 39, 103–109.
A comparative overview of immunoglobulin genes and the generation of their diversity in tetrapods.CrossRef | 22366185PubMed | open url image1

Tarlinton, R., Meers, J., Hanger, J., and Young, P. (2005). Real-time reverse transcriptase PCR for the endogenous koala retrovirus reveals an association between plasma viral load and neoplastic disease in koalas. The Journal of General Virology 86, 783–787.
Real-time reverse transcriptase PCR for the endogenous koala retrovirus reveals an association between plasma viral load and neoplastic disease in koalas.CrossRef | 1:CAS:528:DC%2BD2MXit12lt7k%3D&md5=d127db08cc62a7238562a18fc0e7dd86CAS | 15722540PubMed | open url image1

Thompson, J. D., Higgins, D. G., and Gibson, T. J. (1994). CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position specific gap penalties and weight matrix choice. Nucleic Acids Research 22, 4673–4680.
CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position specific gap penalties and weight matrix choice.CrossRef | 1:CAS:528:DyaK2MXitlSgu74%3D&md5=fe8a0b3b36e19928691eb73390490820CAS | 7984417PubMed | open url image1

Wong, E. S., Young, L. J., Papenfuss, A. T., and Belov, K. (2006). In silico identification of opossum cytokine genes suggests the complexity of the marsupial immune system rivals that of eutherian mammals. Immunome Research 2, 4.
In silico identification of opossum cytokine genes suggests the complexity of the marsupial immune system rivals that of eutherian mammals.CrossRef | 17094811PubMed | open url image1

Wong, E. S. W., Papenfuss, A. T., and Belov, K. (2011). Immunome database for marsupials and monotremes. BMC Immunology 12, 48.
Immunome database for marsupials and monotremes.CrossRef | 1:CAS:528:DC%2BC3MXhtF2gurjN&md5=ead51cd14c1c8d907c54900d98a3808cCAS | open url image1



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