Abstract
Investigation of the immune response of the koala (Phascolarctos cinereus) is needed urgently, but has been limited by scarcity of species-specific reagents and methods for this unique and divergent marsupial. Infectious disease is an important threat to wild populations of koalas; the most widespread and important of these is Chlamydial disease, caused by Chlamydia pecorum and Chlamydia pneumoniae. In addition, koala retrovirus (KoRV), which is of 100% prevalence in northern Australia, has been proposed as an important agent of immune suppression that could explain the koala’s susceptibility to disease. The correct balance of T regulatory, T helper 1 (Th1) and Th2 lymphocyte responses are important to an individual’s susceptibility or resistance to chlamydial infection. The ability to study chlamydial or KoRV pathogenesis, effects of environmental stressors on immunity, and the response of koalas to vaccines under development, by examining the koala’s adaptive response to natural infection or in-vitro stimulation, has been limited to date by a paucity of species- specific reagents. In this study we have used cytokine sequences from four marsupial genomes to identify mRNA sequences for key T regulatory, Th1 and Th2 cytokines interleukin 4 (IL-4), interleukin 6 (IL-6), interleukin 10 (IL-10) and interferon gamma (IFNγ) along with CD4 and CD8β. The koala sequences used for primer design showed >58% homology with grey short-tailed opossum, >71% with tammar wallaby and 78% with Tasmanian devil amino acid sequences. We report the development of real-time RT-PCR assays to measure the expression of these genes in unstimulated cells and after three common mitogen stimulation protocols (phorbol myristate acetate/ionomycin, phorbol myristate acetate/phytohemagglutinin and concanavalin A). Phorbol myristate acetate/ionomycin was found to be the most effective mitogen to up-regulate the production of IL-4, IL-10 and IFNγ. IL-6 production was not consistently up-regulated by any of the protocols. Expression of CD4 and CD8β was down-regulated by mitogen stimulation. We found that the reference genes GAPDH and 28s are valid for normalising cytokine expression by koala lymphocytes after mitogen stimulation.
Highlights
There is a long-standing and growing need to understand the immune system of the koala (Phascolarctos cinereus)
Koala Retrovirus (KoRV) infection occurs at 100% prevalence in northern Australia (New South Wales and Queensland) but has a lower prevalence in the southern states (Victoria and South Australia) (Simmons et al, 2012) and, KoRV has been associated with neoplastic disease in koalas (Tarlinton et al, 2005; Xu et al, 2013) the relationship between KoRV and infectious diseases is so far unclear (Simmons, 2011; Tarlinton et al, 2005)
Of the three candidate reference genes, 28 s and GAPDH were considered suitable for all treatments, with a mean relative expression usually
Summary
There is a long-standing and growing need to understand the immune system of the koala (Phascolarctos cinereus). Immunological studies are key to understanding the pathogenesis of this disease, assessing the effects of stressors on the immunocompetence of koalas, and assessing responses to vaccines currently under development (Carey et al, 2010; Kollipara et al, 2012). They are needed to evaluate the suggestion that Koala Retrovirus (KoRV), is an important agent of immune suppression that may explain koalas’ susceptibility to chlamydial infection (Tarlinton et al, 2005). KoRV infection occurs at 100% prevalence in northern Australia (New South Wales and Queensland) but has a lower prevalence in the southern states (Victoria and South Australia) (Simmons et al, 2012) and, KoRV has been associated with neoplastic disease in koalas (Tarlinton et al, 2005; Xu et al, 2013) the relationship between KoRV and infectious diseases is so far unclear (Simmons, 2011; Tarlinton et al, 2005)
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