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.

The aim of this study was to use real-time polymerase chain reaction assays to determine the prevalence of three haemoplasma species in cats from Greece and to evaluate possible associations between haemoplasma infection and age, gender, feline immunodeficiency virus/feline leukaemia virus (FIV/FeLV) status and packed cell volume (PCV). Ninety-seven cats (24 ill anaemic, 55 ill non-anaemic, 18 healthy non-anaemic) were included in the study. Twenty cats (20.6%) were haemoplasma positive; seven cats were infected only with Mycoplasma haemofelis, 10 were infected only with 'Candidatus Mycoplasma haemominutum' and three were co-infected with M haemofelis and 'Candidatus M haemominutum'. 'Candidatus Mycoplasma turicensis' was not detected. Haemoplasma infection was associated with older age (P=0.019). M haemofelis infection tended to be more common in anaemic cats (P=0.058). No association between gender and haemoplasma infection, or haemoplasma relative copy number and PCV, was detected. Retroviral infection rates were very low with only one FeLV proviral positive cat found.

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.

Koala Retrovirus (KoRV) has been widely speculated to cause immune suppression in koalas ( Phascolarctos cinereus ) and to underlie the koala’s susceptibility to infectious disease, however evidence for immunomodulation is limited. The aim of this study is to determine whether immunophenotypic changes are associated with KoRV infection in free ranging Victorian koalas. qPCR was used to examine mRNA expression for Th1 (IFNγ), Th2-promoting (IL6, IL10) and Th17 (IL17A) cytokines, along with CD4 and CD8 in whole blood of koalas (n = 74) from Mt Eccles and Raymond Island in Victoria, Australia, with and without natural chlamydial infection. KoRV positive koalas had significantly lower levels of IL17A ( p ` 0 . 023 ) and IFNγ ( p = 0 . 044 ) gene expression along with a decreased CD4:CD8 gene expression ratio ( p = 0 . 025 ) compared to negative koalas. No effect of chlamydial infection or combined effect of KoRV and chlamydial infection was detected in these populations. The decreased expression of IFNγ could make KoRV infected koalas more susceptible to persistent chlamydial infection, and a decrease in IL17A could make them more susceptible to gram negative bacterial, fungal and mycobacterial infection; but more tolerant of chlamydial infection.

The effect of 24- and 48-hour storage at room temperature on automated total nucleated cell count (TNCC), differential cell count (DCC) and cell morphology was assessed, and the effect of initial total protein concentration on canine and feline body cavity effusion samples (2 to 5 ml) was evaluated. At 24 and 48 hours, TNCC and absolute numbers of neutrophils, macrophages and small lymphocytes were significantly decreased and numbers of unrecognisable cells were significantly increased. Neoplastic cells and intracellular bacteria identified in fresh samples were missed at 24 and 48 hours. The initial total protein concentration was associated with an effect on percentage of unrecognisable cells and small lymphocytes over time. Change in TNCC over time would have resulted in misclassification of the effusion type in four of 47 samples.