In this study, koalas from two geographically separated populations in SE Qld, at the Moreton Bay site (MB)9 and the Old Hidden Vale site (HV), underwent regular field monitoring and clinical examinations approximately every 6 months (or more frequently if required for health or welfare concerns). Blood samples, ocular conjunctival swabs and a urogenital tract swab were collected during each clinical examination. From these samples, C. pecorum load and genotype, koala MHC immunogenetics and KoRV proviral subtypes were determined. These results were evaluated in the context of clinical records compiled at the time of sample collection, which included chlamydial disease status.
Chlamydial epidemiology at each study site
The overall prevalence of chlamydial infection and disease differed between the study sites
Longitudinal monitoring of 24 HV koalas (over 113 individual sampling points) identified 24 chlamydial infections for strain typing analysis and eight new chlamydial infections for disease progression analysis. This complemented longitudinal monitoring of 148 MB koalas (over 479 individual sampling points)9 that identified 76 chlamydial infections for strain typing analysis and 38 new chlamydial infections for disease progression analysis. Overall, there was a significantly higher prevalence of infection at HV (58%, 14/24) compared to MB (35%, 89/254)26 (Fisher’s exact test p = 0.028) (Table 1), as well as a significantly higher prevalence of disease at HV (58%, 14/24) compared to MB (27%, 75/279) 26 (Fisher’s exact test p = 0.002).
Chlamydial disease progression was common at both study sites
A total of eight HV koalas met our study inclusion criteria for disease progression analysis by having a new chlamydial infection detected at the ocular (n = 1) or urogenital tract site (n = 7) by quantitative polymerase chain reaction (qPCR) over a period of 18 months. These koalas had no evidence of chlamydial infection (infection loads below detection limit) or disease (clinical examination within normal limits) at that anatomical site at their previous clinical examination. If disease was detected at their first clinical examination, they were excluded from disease progression analyses only (unless it was their first sampling as an independent offspring, n = 1).
Interestingly, all of the new chlamydial infections at HV (100%, 8/8) progressed to disease, which was not significantly different to the number of new chlamydial infections at MB that progressed to disease (66%, 25/38)9 (Fisher’s exact test p = 0.084) (Supplementary Fig. S1). For six of these new chlamydial infections at HV (one ocular and five urogenital tract), the infection was detected at the same clinical examination as disease. For the other two new chlamydial infections at HV (both urogenital tract), the infection was present at a clinical examination 2.5 months and 4 months before disease was detected.
The urogenital tract infection load dynamics were similar at both study sites
The urogenital tract infection load (C. pecorum genome copies/µL) in both HV and MB9 koalas was significantly higher when infections were detected at the same clinical examination as disease (1,028,000 copies/µL, range 11,400–4,760,000 copies/µL), in comparison to infections that were present for one or more consecutive clinical examinations before disease was detected or infections that did not progress to disease (600 copies/µL, range 49–522,800 copies/µL) (Mann–Whitney U = 3, p = 0.030). Similarly, the urogenital tract infection load in both HV and MB9 koalas was significantly higher when koalas acquired a new chlamydial infection (within the last three months) (1,834,000 copies/µL, range 52,400–4,760,000 copies/µL), compared to koalas who had long-term infections (present for more than three months) (724 copies/µL, range 35–7,142 copies/µL) (Mann–Whitney U = 0, p = 0.010). Interestingly, the urogenital tract infection load was significantly higher at HV (1,028,000 copies/µL, range 11,400–4,760,000 copies/µL) compared to MB (3,824 copies/µL, range 138–1,340,000 copies/µL) when infections were detected at the same clinical examination as disease (Mann–Whitney U = 11, p = 0.003). In contrast, the urogenital tract infection load was not significantly different between the study sites (HV 600 copies/µL, range 49–522,800 copies/µL vs MB 794 copies/µL, range 16–13,900 copies/µL) when infections were present for one or more consecutive clinical examinations before disease was detected or infections did not progress to disease (Mann–Whitney U = 28, p = 0.703).
The prevalence of chlamydial strains, as determined by Multi-Locus Sequence Typing, differed between the study sites
Overall, 69 C. pecorum-positive samples, comprised of 45 samples from MB (4 ocular and 41 urogenital tract samples from 25 koalas) and 24 samples from HV (2 ocular and 22 urogenital tract samples from 14 koalas), were analysed using a C. pecorum-specific MLST scheme27. Three sequence types (STs) were detected in this study: ST 69, ST 202 and a novel ST (ST 281). ST 69 and ST 202 were detected at both study sites, however their prevalence at each study site was significantly different (Fig. 1). ST 69 was the most prevalent ST at HV, detected in 63% of total samples (15/24) and in 59% of urogenital tract site samples (13/22). ST 69 was significantly less prevalent at MB, detected in 11% of total samples (5/45) and in 12% of urogenital tract site samples (5/41) (overall and urogenital tract site Fisher’s exact test p < 0.001). In contrast, ST 202 was the most prevalent ST at MB, detected in 87% of total samples (39/45) and in 88% of urogenital tract site samples (36/41). ST 202 was significantly less prevalent at HV, detected in 13% of total samples (3/24) and in 14% of urogenital tract site samples (3/22) (overall and urogenital tract site Fisher’s exact test p < 0.001). The novel ST at HV, ST 281, was detected at the urogenital tract site in three koalas, representing 25% of total samples (6/24) and 27% of urogenital tract site samples (6/22). In comparison to the other previously described koala C. pecorum STs, ST 202 clustered in a well-supported, closely related clade with STs commonly detected in Qld and NSW koalas, including ST 220, ST 199 and ST 70 (Supplementary Fig. S2). ST 69 and ST 281, however, clustered into their own clades.
Mid-point rooted Bayesian phylogenetic trees constructed using the concatenated Multi-Locus Sequence Typing alignment (ST) (left) and ompA alignment (right) detected at the Moreton Bay site (MB—in red) and the Old Hidden Vale site (HV—in blue) (K# denotes koala number, U or O for urogenital vs ocular sample, T# for timepoint from longitudinal samples, clinical groups are marked with coloured lines; black denotes the resolver group, pink denotes the chronic infection group, green denotes the diseased after chronic infection group, red denotes the infected and diseased group, yellow denotes the diseased at first exam group), E58 is an ompA sequence fragment that is identical to the bovine E58 strain (created using Inkscape 0.92.3, https://inkscape.org/).
The prevalence and diversity of chlamydial strains, as determined by ompA genotyping, differed between the study sites
To compare ompA genotypes between MB and HV, all 24 C. pecorum-positive HV samples were genotyped and compared to previously reported MB genotypes (62 total samples, 12 ocular and 50 urogenital tract samples)9 (Fig. 1). The ompA genotype F was the most prevalent ompA genotype at HV, detected in 79% of total samples (19/24) and in 82% of urogenital tract site samples (18/22). The ompA genotype F was significantly less prevalent at MB, detected in 10% of total samples (6/62) and in 12% of urogenital tract site samples (6/50)9 (overall and urogenital tract site Fisher’s exact test p < 0.001). In contrast, the ompA genotype E´ was the most prevalent ompA genotype at MB, detected in 60% of total samples (37/62) and in 64% of urogenital tract site samples (32/50)9. The ompA genotype E´ was significantly less prevalent at HV and was the second most prevalent ompA genotype at this study site, detected in 17% of total samples (4/24) and in 18% of urogenital tract site samples (4/22) (overall and urogenital tract site Fisher’s exact test p < 0.001). The only other ompA genotype detected at HV was the ompA genotype A´, which was detected in a single ocular site sample.
Although a diverse range of ompA genotypes was detected at the urogenital tract site at MB, including A´, E´, F, F´, G and an ompA sequence fragment identical to that of the bovine E58 strain9, only ompA genotypes F and E´ were detected at the urogenital tract site at HV (Supplementary Fig. S2). For multifocal infections (ocular and urogenital tract sites) at HV (n = 2), the ompA genotype detected at each anatomical site differed. In one case, ompA genotype A´ was detected at the ocular site and ompA genotype F was detected at the urogenital tract site, and in the other, ompA genotype F was detected at the ocular site and ompA genotype E´ was detected at the urogenital tract site. Overall, the diversity in ompA, where six genotypes were characterised, was higher than the diversity in STs, where only three STs were characterised (Fig. 1). The C. pecorum plasmid was detected in all chlamydial strains except for a single ocular site sample from one koala at MB (see Supplementary Information for more details).
The dynamics of chlamydial strains during long-term infections differed between the strain typing methods
A total of 10 long-term urogenital tract infections were analysed with both strain typing methods, three at HV and seven at MB (Fig. 2). Although identical STs were detected over time in all long-term urogenital tract infections (100%, 10/10), genetically distinct ompA genotypes were detected over time in three of these long-term urogenital tract infections (30%, 3/10) at MB9. ST 202 was always detected in these infections, however the ompA genotype changed from F to A´ (4.5 months later) in one case, G to E´ (12 months later) in another case and G to E´ (7 months later) and again to A´ (10.5 months later) in the last case. An additional six ompA sequences from ocular (n = 1) and urogenital tract site (n = 5) samples collected from MB koalas, not previously reported by us9, were included in this analysis. Unfortunately, although the ompA fragment was amplified in a further two ocular and six urogenital tract site samples, we were not able to resolve the ompA sequences from these chromatograms.
Schematic of long-term infections over time, demonstrating changing ompA genotypes but identical Multi-Locus Sequence Typing sequence types (STs) (boxes denote sampling points and the time between sampling points is indicated in the top scale bar) (created using Inkscape 0.92.3, https://inkscape.org/).
Impact of chlamydial genotype on disease progression
Urogenital tract disease progression at each study site was associated with the chlamydial strain
At MB, ST 69 was significantly more prevalent in koalas that resolved their infections without progressing to disease (60%, 3/5) (the resolver group) compared to koalas that did not resolve their infections (6%, 2/36) (the chronic infection, the diseased after chronic infection and the infected and diseased groups combined) (Fisher’s exact test p = 0.009). Interestingly, at HV, ST 281 was significantly more prevalent in koalas that had not progressed to disease (56%, 5/9) (the chronic infection group) compared to koalas that had progressed to disease (1/12, 8%) (the infected and diseased and the diseased after chronic infection groups combined) (Fisher’s exact test p = 0.046). Further, when STs and ompA genotypes were concatenated to form a Multi-Locus Sequence Analysis (MLSA) type, MLSA type 2 (ST 281 with ompA genotype F) was significantly more prevalent in koalas that had not progressed to disease (56%, 5/9) compared to koalas that had progressed to disease (8%, 1/12) (Fisher’s exact test p = 0.046). In contrast, MLSA type 1 (ST 69 with ompA genotype F) was significantly more prevalent in koalas that had progressed to disease (75%, 9/12) compared to koalas that had not progressed to disease (22%, 2/9) (Fisher’s exact test p = 0.030).
When urogenital tract disease progression and chlamydial strains were analysed across both study sites combined (41 MB and 21 HV koalas for STs, 35 MB and 21 HV koalas for ompA genotypes and MLSA types), there were no significant differences in the prevalence of STs, ompA genotypes or MLSA types in koalas that had progressed to disease compared to koalas that had not progressed to disease.
Immunogenetic profiles at each study site
The major histocompatibility complex haplotypes were genetically diverse between the study sites
To investigate MHC gene diversity at each site, 60 koalas were selected from MB (30 koalas from our previous chlamydial epidemiology analyses (see Robbins et al.9 for more details) and 30 additional healthy koalas) and 20 koalas were selected from HV (sexually mature koalas undergoing monitoring). The MHC allele diversity in two Class I genes (UA and UC) and four Class II genes (DAb, DBb, DMb and DCb) was determined (Supplementary Fig. S3).
Overall, MHC haplotypes clustered based on study site (Fig. 3). Koalas grouped into three MHC haplotype clusters based on more than 65% genetic similarity. Two of these clusters represented MB koalas while the third cluster represented HV koalas. There was a tendency for MB koalas to belong to the same MHC cluster when they were captured from a similar geographical location within the 13 km site, suggesting some geographical sub-population structure. MB clusters 1 and 2 overlapped, however, and there are records of both natural dispersal and koala translocations within this study site. Interestingly, despite the separation of the MB and HV koalas by the BVB23,24 (and approximately 70 kms), three MB koalas genetically grouped within HV cluster 3 while two HV koalas genetically grouped within MB cluster 1.
(a) Cluster dendrogram of major histocompatibility complex (MHC) haplotypes indicating clustering by study site but not by disease progression (red line denotes > 65% genetically identical, red squares denote Moreton Bay site (MB), blue squares denote Old Hidden Vale site (HV), green squares denote developed disease during monitoring, orange squares denote did not develop disease during monitoring) (b) Schematic showing study sites and capture location of koalas within MHC haplotype clusters (dashed line denotes the Brisbane Valley biogeographical barrier) (created using Inkscape 0.92.3, https://inkscape.org/).
The prevalence of major histocompatibility complex alleles differed between the study sites
Overall, 61 MHC alleles were detected in this study (Supplementary Fig. S3). In total, 21 MHC alleles were only detected at MB, 14 MHC alleles were only detected at HV and 26 MHC alleles were detected at both study sites. There were eight MHC alleles that had a significantly higher prevalence at each study site (Fisher’s exact test p < 0.05), with UA 10:01, UA 14:01, UC 01:01, UC 05:02, DAb 10, DAb 19, DBb 03 and DMb 04 significantly more prevalent in MB koalas and UA 08:01, UA 11:01, UA 17:01, UC 01:03, DAb 22, DAb 23, DAb 37 and DBb 04 significantly more prevalent in HV koalas (Supplementary Table S1). In addition, this study expanded our knowledge of koala MHC allele diversity, with a total of 28 previously unreported MHC alleles being identified (15 at MB, 12 at HV and one at both study sites) (Supplementary Fig. S3).
Impact of host genetics on disease progression
Urogenital tract disease progression was associated with major histocompatibility complex alleles
At each study site, the prevalence of only one individual MHC allele was significantly different between koalas that progressed to disease and koalas that did not progress to disease. At MB, the Class II allele DAb 10 was significantly more prevalent in koalas that did not progress to disease (50%, 17/34) compared to koalas that progressed to disease (13%, 3/23) (Fisher’s exact test p = 0.005). In contrast, at HV, the Class II allele DCb 03 was significantly more prevalent in koalas that progressed to disease (75%, 9/12) compared to koalas that did not progress to disease (14%, 1/7) (Fisher’s exact test p = 0.020).
When urogenital tract disease progression and koala immunogenetics were analysed across both study sites combined (57 MB and 19 HV koalas), the prevalence of four individual MHC alleles was significantly different between koalas that progressed to disease and koalas that did not progress to disease. The Class II allele DCb 03 was significantly more prevalent in koalas that progressed to disease (66%, 23/35) compared to koalas that did not progress to disease (34%, 14/41) (Fisher’s exact test p = 0.011), and koalas with DCb allele 03 were 3.70 times (95% CI 1.43–9.56 times) more likely to progress to disease. The Class II allele DBb 04 was also significantly more prevalent in koalas that progressed to disease (11%, 4/35) compared to koalas that did not progress to disease (0%, 0/41) (Fisher’s exact test p = 0.041). In contrast, the Class II allele DAb 10 was significantly more prevalent in koalas that did not progress to disease (42%, 17/41) compared to koalas that progressed to disease (11%, 4/35) (Fisher’s exact test p = 0.004), and koalas with DAb allele 10 were 5.49 times (95% CI 1.63–18.46 times) less likely to progress to disease. Finally, the Class I allele UC 01:01 was also significantly more prevalent in koalas that did not progress to disease (100%, 41/41) compared to koalas that progressed to disease (89%, 31/35) (Fisher’s exact test p = 0.041).
MHC haplotypes were not, however, associated with overall disease progression, with koalas that progressed to disease at any anatomical site at any time during the period of monitoring distributed evenly throughout the genetic clusters (Fig. 3).
Koala retrovirus profiles at each study site
The prevalence of most koala retrovirus subtypes, except KoRV-F, was similar between the study sites
To investigate the KoRV profiles of koalas at each study site, the KoRV proviral env gene was amplified and sequenced for the same 80 koalas used for the MHC gene diversity analysis (60 MB koalas over 68 individual sampling points and 20 HV koalas over 22 individual sampling points). Overall, five KoRV subtypes were detected, with KoRV subtypes -A, -B, -D and -F detected at both study sites and KoRV-G only detected at MB.
When only the first time-point was included for koalas with longitudinal samples, KoRV-F was significantly more prevalent in MB koalas (77%, 46/60) compared to HV koalas (25%, 5/20) (Fisher’s exact test, p < 0.001) (Supplementary Fig. S4. There were no significant differences in the prevalence of any other KoRV subtype between the study sites. As expected, KoRV-A prevalence was 100% in both MB koalas (60/60) and HV koalas (20/20). The prevalence of KoRV-B was 40% (24/60) in MB koalas compared to 55% (11/20) in HV koalas (Fisher’s exact test p = 0.301), the prevalence of KoRV-D was 97% (58/60) in MB koalas compared to 100% (20/20) in HV koalas (Fisher’s exact test p = 1.000), and the prevalence of KoRV-G was 2% (1/60) in MB koalas compared to 0% (0/20) in HV koalas (Fisher’s exact test p = 1.000).
Impact of co-infection with koala retrovirus on disease progression
Urogenital tract disease progression was not associated with koala retrovirus profiles
There were no significant differences in the prevalence, proportional abundance or diversity (number of OTUs per koala) of any KoRV subtype between koalas that had progressed to disease (the infected and diseased and the diseased after chronic infection groups combined) and koalas that had not progressed to disease (the chronic infection group) at either study site (35 MB and 13 HV koalas), or when urogenital tract disease progression and KoRV profiles were analysed across both study sites combined.
Source: Ecology - nature.com
