Elsevier

Veterinary Microbiology

Volume 231, April 2019, Pages 264-270
Veterinary Microbiology

Genetic differences in Chlamydia pecorum between neighbouring sub-populations of koalas (Phascolarctos cinereus)

https://doi.org/10.1016/j.vetmic.2019.02.020Get rights and content

Highlights

  • Differing disease expressions were associated with different MLST sequence types.

  • Differences in MLST sequence types suggests multiple strain emergence events.

  • Identical MLST sequence types occur across the koalas range.

  • The markers used in the MLST scheme may not be reflective of strain pathogenicity.

Abstract

Chlamydiosis, caused by Chlamydia pecorum, is regarded as an important threat to koala populations. Across the koala’s geographical range, disease severity associated with C. pecorum infection varies, with pathogen diversity and strain pathogenicity being likely important factors. To examine C. pecorum diversity on a sub-population level a Multi-Locus Sequence Typing (MLST) scheme, containing the housekeeping genes; gatA, oppA_3, hflX, gidA, enoA, hemN and fumC, was used to type strains from two sub-populations of koalas from the Liverpool Plains, NSW, Australia, with different disease expressions. Typing of samples from 2015 to 2017, revealed a significant association between sequence type ST 69 and clinical disease and a significant difference in sequence type frequencies between sub-populations. Sequence type ST 69 has previously been identified in both subclinical and clinically diseased koalas indicating that these markers alone are not illustrative of pathogenicity. However, recent emergence of this sequence type in a naïve population may explain the differing disease expressions. Sequence types ST 73 and ST 69 have been described in koalas across a broad geographic range, indicating multiple introduction events and/or a limited veracity of the MLST loci to explore fine scale epidemiological investigations, particularly those examining the interface between pathogenic strain and disease outcome.

Introduction

Chlamydiosis is the leading cause of keratoconjunctivitis (Brown and Grice, 1984; Cockram and Jackson, 1981), urogenital disease and infertility in the koala (Brown and Grice, 1984; McColl et al., 1984), with Chlamydia pecorum being the main causative chlamydial species (Jackson et al., 1999). It is transmitted predominantly venereally but can also be transmitted vertically from mother to young (Jackson et al., 1999; Nyari et al., 2017; Russell et al., 2018).

Although this pathogen is sometimes regarded as an important driver of population declines (Griffith et al., 2013; Rhodes et al., 2011) there is little direct evidence demonstrating this, and the impact of chlamydiosis across most koala populations in Australia remains poorly understood (McCallum et al., 2017). Among infected koalas subclinical chlamydial disease is common (Jackson et al., 1999; Nyari et al., 2017) and koalas with clinical signs display varying degrees of severity (Wan et al., 2011; Weigler et al., 1988). Differences in disease expression are observable across koala populations throughout Australia (Kollipara et al., 2013; Legione et al., 2016; Patterson et al., 2015; Polkinghorne et al., 2013) with the factors driving these variabilities being multifactorial and complex. Such factors include the immunological profile of individuals (Mathew et al., 2014), co-infection with other pathogens such as koala retrovirus (Waugh et al., 2017) and differences in C. pecorum strains. Genetic variation has been reported amongst strains isolated from different regions (Jackson et al., 1997; Jelocnik et al., 2013; Kollipara et al., 2013; Legione et al., 2016; Marsh et al., 2011) with it being suggested that C. pecorum strains may likely differ in virulence (Higgins et al., 2012).

Improved understanding of pathogen diversity and significance within populations is needed to refine the knowledge on the epidemiology of C. pecorum infections and evaluate the risks of pathogen transfer when managing translocations and constructing wildlife corridors between koala subpopulations.

Meta-analysis of C. pecorum diversity in koalas is challenging because of the variation in methods and gene targets used in different studies. Single-locus typing of the ompA gene coding for the major outer membrane protein has been commonly applied (Jackson et al., 1997; Kollipara et al., 2013). Yet the accuracy of using this gene alone to infer relatedness and biogeographic spread of strains is considered suboptimal compared to its use in combination with other loci (Marsh et al., 2011). Whole genome sequencing (WGS) has been used to characterise varying regions within the C. pecorum genome (Bachmann et al., 2014). However, the high cost of applying this method makes it impractical when dealing with large sample sizes. Thus, there is a need for a reliable typing method with sufficient resolution to characterize strains and to be applicable across various typing studies. Addressing the limitations of single locus typing and whole genome analysis, Multi-Locus Sequence Typing (MLST) has been applied as a useful typing tool with sufficient sensitivity to elucidate the diversity of strains (Maiden, 2006). MLST types strains based on housekeeping genes that have been demonstrated to be useful markers due to their highly conserved nature (Eisenberg and Levanon, 2013; Feil, 2004; Maiden, 2006; Martin et al., 1998). In previous typing studies of C. trachomatis, C. pneumoniae and C. pecorum, MLST has suggested the potential for sequence types (ST) to be associated with certain clinical outcomes (Jelocnik et al., 2014; Pannekoek et al., 2008). Current typing studies of C. pecorum within Australia have centred on highly conserved housekeeping genes applied to limited sample sizes of koalas (Jelocnik et al., 2013, 2014). The application of MLST to a greater sample size of koala isolates is required to better understand both intra- and inter-population strain diversity (Jelocnik et al., 2013).

The Gunnedah Shire (30° 59′ S, 150° 16′ E) is situated within the Liverpool Plains, northern NSW, Australia (Fig. 1) and is home to a large number of free-ranging koala sub-populations in fragmented habitats (Crowther et al., 2014; Lunney et al., 2012). The emergence of chlamydiosis in the Gunnedah regional koala population, with distinct sub-population variation in disease expression over a 10-year period (M. Krockenberger, 2017, personal observation) offered an ideal clinical scenario to test the value of MLST for fine scale epidemiology of disease emergence of koala chlamydiosis. In addition, it also allowed investigation of the association of MLST strain variation in C. pecorum with disease outcomes in two apparently discontinuous koala sub-populations.

Section snippets

Ethics statement

All handling and sampling of koalas was conducted by experienced veterinarians under the University of Sydney Animal Ethics Approval Number 2016/955 and NPWS Scientific License SL101687.

Animals included from the Gunnedah Shire and disease severity

Samples and data were from 140 koalas captured in 2015 (n = 24), 2016 (n = 73) and 2017 (n = 43) with 68 specimens from the southeast sub-population and 72 from the northwest sub-population (Table 2) (Fig. 1). Three clinical swabs were collected per koala: two ocular swabs from the conjunctiva of the left and

Local epidemiology and sequence type diversity

Between 2015 and 2017, specimens from 140 koalas were collected and screened for the presence of Chlamydia with 90/140 (64%) being positive for C. pecorum at the urogenital site (Table 2). Of the 34/140 (24%) koalas positive for C. pecorum at the ocular site, 31/140 (22%) were also positive at the urogenital site. The proportion of samples from the southeast sub-population with C. pecorum at the ocular 20/68 (29%) and urogenital sites 58/68 (85%) from 2015 to 2017 was comparatively higher than

Discussion

Two sub-populations of koalas in the Gunnedah shire in the Liverpool Plains which differed in disease prevalence and severity, had different associated chlamydial sequence type patterns. In this region, ST 69 was described in only one of the two koala sub-populations (the northwest sub-population), in association with clinical disease at the urogenital and ocular regions. In contrast, ST 73 was found across both sub-populations, where it was associated with a higher apparent prevalence and

Conclusion

The inclusion of two sub-populations that display different disease expression, while sharing similar habitats, provided a model to explore the diversity of C. pecorum and its role in disease severity at a population level. Significant differences in the frequency of sequence types ST 73 and ST 69 between the two sub-populations, has demonstrated that differences in C. pecorum strains can and do exist between neighbouring populations from within the same region, suggesting multiple disease

Funding

Field work expenses were funded by the Australian Research Council (ARC) Linkage Project Grant LP140100279, with OEH and Shenhua Australia Holdings as linkage partners.

Conflicts of interest

The author acknowledges no conflicts of interest.

Declarations of interest

None.

Acknowledgements

We are indebted to George Madani for his skill in catching koalas in the wild. A large number of staff and honours students and PhD students and volunteers from The University of Sydney and the Office of Environment and Heritage (OEH) provided assistance with the sample collection over many years, including Dan Lunney, Clare McArthur, Joanna Griffith, Caroline Marschner, Adrian Rus, Alina Kelman, Chandra Diamant, Alex Kan amongst others. OEH staff, including Lachlan Wilmott, John Lemon and Rob

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    Present address: Sydney School of Veterinary Science, The University of Sydney, Sydney, 2006, NSW, Australia.

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