PATHOGENESIS OF CHLAMYDIOSIS

A detailed understanding of the pathogenesis of chlamydiosis in koalas is lacking. Nevertheless, it is recognised that chlamydiosis in koalas shares all the hall­marks of the disease in other species; namely, scarring and fibrosis of mucosal tissues during and following infection.

Chlamydia spp. encode an arsenal of virulence factors, including a cytotoxin, membrane attack complex/per- forin protein (MACPF), phospholipase D, secreted effec­tors, polymorphic membrane proteins, heat shock proteins (HSPs) and virulence plasmid proteins (Bach­mann et al. 2014b). These factors enable Chlamydiae to exploit numerous host cellular signalling, cytoskeletal and membrane pathways to infect host cells, complete their developmental cycle and ensure their own survival by inhibiting phagocytosis and apoptosis, activating host cell pro-survival pathways and avoiding the host immune system (Elwell et al. 2016; Murthy et al. 2016). Compara­tive genomic analyses have shown C. pecorum exhibits all of these virulence factors (e.g. cytotoxin gene, virulence plasmid, secreted effectors) and that subtle genetic differ­ences, noted as single nucleotide polymorphisms, may influence pathogenicity and tissue tropism in koala strains (Bachmann et al. 2014a). The virulence plasmid occurs less frequently in strains infecting koalas in SA (which generally have mild or subclinical disease) than in strains infecting koalas in Qld and NSW (which have a higher prevalence of overt clinical disease) (Jelocnik et al.

2015). As there is strong evidence linking the chlamydial plasmid to pathogenicity in other chlamydial species (Zhong 2017), variable plasmid distribution in koala strains may explain differences in the prevalence of clini­cal disease.

Although genetic differences in the C. pecorum strains infecting koalas may offer an explanation for variable virulence, studies of chlamydial disease in humans and in other animal models have shown that host immunologi­cal response is a critical factor in the development of chlamydial disease (Elwell et al. 2016; Murthy et al. 2016). Chlamydiae can infect a range of epithelial, endothelial and immune cells, such as macrophages, by binding to a range of host cell receptors. Epithelial and endothelial Chlamydia-infected cells produce a range of proinflam- matory cytokines and chemokines (such as interferon­gamma (IFNγ), interleukins (IL) and tumour necrosis factor-alpha [TNF-α]), in addition to matrix metallopro­teases (MMPs) and neutrophils recruited to the site, which may lead to subsequent tissue damage.

Chlamydial persistence, whereby the pathogen evades the host immune responses by remaining in a non- replicative intracellular state, may also lead to continuing damage to the host (Elwell et al. 2016; Murthy et al. 2016). During persistence, Chlamydiae can produce highly con­served HSPs to preserve cellular activity. Chlamydial HSPs can molecularly mimic human endogenous pro­teins (Elwell et al. 2016; Murthy et al. 2016). Immune cells (primarily T-cells) and antibody responses appear to be able to target these chlamydial HSPs, thereby contribut­ing to tissue damage and sequelae in the host. Repeated episodes of infection with Chlamydia in children can lead to severe conjunctival inflammation, scarring and sustained immune response, leading to potentially blind­ing trichiasis later in life (Taylor et al. 2014).

At present, little is known of the koala host response to chlamydial infection and the role of the host immune response in development of clinical disease. There are few koala-specific immunological assays available to investi­gate this process. The recent availability of koala genomic (https://koalagenome.org/) and transcriptomic resources and cytokine assays has provided insight into the koala immune response and allowed characterisation of koala immune genes (Australian Museum and Koala Genome Consortium 2014; Hobbs et al.

In terms of a cellular response, natural killer (NK) cells are a key part of the mammalian innate and adaptive immune responses against chlamydial infection. Recent characterisation of genes encoding koala NK cells and their receptors provided evidence that these genes may be upregulated in koalas with Chlamydia, suggesting these cells may also play a role in the immune response (Morris et al. 2015). Similar to human chlamydial-associated infertility, chlamydial HSPs (c-HSP10 and c-HSP60) were associated with fibrous occlusion of the koala uterus and oviduct (Higgins et al. 2005). High titres of serum immu­noglobulin G against these proteins have been repeatedly observed in koalas with chlamydial disease (Higgins et al. 2005; Lau et al. 2014). Genetic studies in other species suggest that some genetic factors may predispose indi­viduals to Chlamydia-associated scarring, but this has not been evaluated in koalas.

Pagliarani et al. (2022) examined the pathogenesis of the most common pathological lesion associated with chlamydiosis in female koalas, the cystic dilation of the ovarian bursa starting from the evidence that Chlamydia spp.

4.