Altered Immune Cytokine Expression Associated with KoRV B Infection and Season in Captive Koalas

Abstract

Koala (Phascolarctos cinereus) populations are increasingly vulnerable and one of the main threats is chlamydial infection. Koala retrovirus (KoRV) has been proposed as an underlying cause of the koala’s susceptibility to infection with Chlamydia and high rates of lymphoid neoplasia; however, the regionally ubiquitous, endogenous nature of this virus suggests that KoRV A infection is not sufficient for immune suppression to occur. A recently discovered exogenous variant of KoRV, KoRV B, has several structural elements that cause increased pathogenicity in related retroviruses and was associated with lymphoid neoplasia in one study. The present study assesses whether KoRV B infection is associated with alterations in immune function. Cytokine gene expression by mitogen stimulated lymphocytes of KoRV B positive (n = 5–6) and negative (n = 6–7) captive koalas was evaluated by qPCR four times (April 2014-February 2015) to control for seasonal variation. Key immune genes in the Th1 pathway (IFNγ, TNFα), Th2 pathway (IL 10, IL4, IL6) and Th17 pathway (IL17A), along with CD4:CD8 ratio, were assessed. KoRV B positive koalas showed significantly increased up-regulation of IL17A and IL10 in three out of four sampling periods and IFNγ, IL6, IL4 and TNFα in two out of four. IL17A is an immune marker for chlamydial pathogenesis in the koala; increased expression of IL17A in KoRV B positive koalas, and concurrent immune dysregulation, may explain the differences in susceptibility to chlamydial infection and severity of disease seen between individuals and populations. There was also marked seasonal variation in up-regulation for most of the cytokines and the CD4:CD8 ratio. The up-regulation in both Th1 and Th2 cytokines mirrors changes associated with immune dysregulation in humans and felids as a result of retroviral infections. This is the first report of altered immune expression in koalas infected by an exogenous variant of KoRV and also the first report of seasonal variation in cytokine up-regulation and CD4:CD8 ratio in marsupials.

Differential and defective expression of Koala Retrovirus indicate complexity of host and virus evolution

“…KoRV was originally identified during investigations into the high rates of lymphoid neoplasia (lymphoma and leukaemia) in Queensland koalas 13 . Koalas with lymphoid neoplasia have significantly higher KoRV viral loads in plasma 18 and some strains of KoRV also perturb the cytokine response profile of koala lymphocytes 20 .…”

Assessing the significance of endemic disease in conservation—koalas, chlamydia, and koala retrovirus as a case study

“…A study of Victorian koalas did not show a significant link between KoRV infection and chlamydial disease, but only detected the KoRV‐A subtype in the population (Legione et al., ). A recent study has linked the exogenous KoRV‐B subtype with alterations in koala immune function (Maher & Higgins, ); this result is supported by a reported association between KoRV‐B infection and clinical chlamydial disease in a population of koalas in Queensland (Waugh et al., ); however, this association was based on only 36 animals from a population with a limited geographic distribution; work is likely required to further explore this relationship.…”

Identification of stable reference genes for quantitative PCR in koalas

“…ACTB and GAPDH , the most commonly used reference genes for normalization of quantitative expression studies in koalas 29,37,38 were not ranked as the most stable genes with any of the algorithms or in any experimental group. The BestKeeper algorithm chose ACTB as the second most stable gene in all experimental conditions but in other algorithms and the final comprehensive analysis, its ranking position indicated lower stability.…”