Molecular characterisation and expression analysis of interferon gamma in response to natural Chlamydia infection in the koala, Phascolarctos cinereus

Abstract

Interferon gamma (IFNγ) is a key Th1 cytokine, with a principal role in the immune response against intracellular organisms such as Chlamydia. Along with being responsible for significant morbidity in human populations, Chlamydia is also responsible for wide spread infection and disease in many animal hosts, with reports that many Australian koala subpopulations are endemically infected. An understanding of the role played by IFNγ in koala chlamydial diseases is important for the establishment of better prophylactic and therapeutic approaches against chlamydial infection in this host. A limited number of IFNγ sequences have been published from marsupials and no immune reagents to measure expression have been developed. Through preliminary analysis of the koala transcriptome, we have identified the full coding sequence of the koala IFNγ gene. Transcripts were identified in spleen and lymph node tissue samples. Phylogenetic analysis demonstrated that koala IFNγ is closely related to other marsupial IFNγ sequences and more distantly related to eutherian mammals. To begin to characterise the role of this important cytokine in the koala's response to chlamydial infection, we developed a quantitative real time PCR assay and applied it to a small cohort of koalas with and without active chlamydial disease, revealing significant differences in expression patterns between the groups. Description of the IFNγ sequence from the koala will not only assist in understanding this species' response to its most important pathogen but will also provide further insight into the evolution of the marsupial immune system.

Keywords: CD; Chlamydia; Cluster Differentiation; Complementary DNA; Ct; Evolution; GAPDH; Glyceraldehyde 3-Phosphate Dehydrogenase; IFNγ; IL; Immune system; Interferon gamma; Interleukin; Koala; MHC; MOMP; Major Histocompatibility Complex; Major Outer Membrane Protein; NK; Natural Killer; PBMC; Peripheral Blood Mononuclear Cell; Quantitative Real Time Polymerase Chain Reaction; T helper; TNFα; Th; Threshold Cycle; Tumour Necrosis Factor alpha; UTR; UV; Ultraviolet; Untranslated Region; cDNA; qrtPCR.