Ethics
All handling and sampling of koalas was conducted by experienced veterinarians under the University of Sydney Animal Ethics Approval Number 2019/1547 and NPWS Scientific Licence SL102331. all methods were performed in accordance with the relevant guidelines and regulations.
Cohort recruitment
Koalas recruited to this study were captured within a 15 kilometre radius from − 31.159855, 150.118456, south-west of the town of Gunnedah, New South Wales, using the ‘noose and flag’ technique28. Alfaxalone (Jurox), was used to sedate the koalas at a dose rate of 1.8 mg/kg by intramuscular injection and then oxygen and isoflurane was administered to effect through a fitted mask to anaesthetise the koala. The same veterinarian (SJS) assessed and sampled all koalas. At initial assessment, koalas were classified as either diseased or non-diseased, based on external signs of chlamydiosis, (presence of a “wet bottom” or ocular disease)29. These criteria were chosen as these clinical signs can improve with veterinary treatment30 and infection status of individuals was unknown at initial capture. Following this classification, the koala was randomly assigned to one of two treatment groups, vaccination or placebo. The veterinarian who examined the koalas and administered the treatment was blinded to treatment group allocation. VHF collars (transmitter model M3420, ATS Australia) were fitted to the koalas so they could be radio-tracked and visually monitored from a distance at 2-month intervals throughout the study, and recaptured and clinically evaluated at 6, 12 and 18 months following vaccination.
Koala Clinical Examination
The following procedures were performed at timepoint 0 (recruitment), 6, 12 and 18 months post vaccination. Digital images were taken of the koala’s identification tag, eyes, teeth (for age estimation)31 and perineal region. Koalas were weighed and a body condition score (BCS) ranging from 1–5 was recorded32. Both eyes were assessed for ocular discharge, conjunctival proliferation, and conjunctival chemosis, each criterion with a score between 0 and 3 allocated to each eye29. The perineal region was examined for urine leakage, erythema, or the presence of purulent discharge. A wet bottom score was allocated, based on a modification of the Griffith (2010)29 scoring system, whereby only scores 0–6 were used because scores 7–9 required prolonged observation, not amenable to field examinations. Ultrasonography was performed with the koala in dorsal recumbency33, 34, 35. For ultrasonographic examination of the kidneys, a small patch of fur was clipped on the abdomen ventral to the approximate site of each kidney. Both kidneys were identified and the presence of any structural abnormalities such as dilated renal pelvis, dilated ureters or presence of renal calculi were recorded. For females, the transducer was placed inside the pouch and female reproductive structures visualised by scanning the region cranio-lateral to the bladder. If paraovarian cysts were identified, it was noted if the cyst/s were unilateral, bilateral, multiloculated or singular. A paraovarian cyst score was allocated with 0 = no abnormalities, 1 = unilateral paraovarian cysts identified and 2 = bilateral paraovarian cysts identified. If paraovarian cysts were not detected, a thorough scan of the caudal abdomen was performed to confirm their absence.
Swabs for detection of C. pecorum DNA were collected from the conjunctiva bilaterally and the female urogenital sinus or male urethra (Copan, Interpath, 160C). For conjunctival sampling, swabs were placed into the conjunctival fornix and rotated five times. In females, swabs were inserted 2–3 cm into the urogenital sinus and rotated five times. In males, the penis was everted, and the swab placed 2 cm into the urethra and rotated five times. All swabs were stored at -20°C until processing.
Vaccination
Four peptides from C. pecorum MOMP, were used as the antigens within the vaccine, P1 H-EGMSGDPCDPCATW-OH, P2 H-INYHEWQVGAALSYRLNMLIP-OH, utilised previously13, P3 H-VLQIVSLQINKLKSRKACG-OH, P4 H-KKLLKSAFLSAAFFAG-OH, representative of the fifth conserved region and the leader sequence of MOMP, respectively. All peptides were synthetised by Mimotopes (Melbourne, Australia), at a purity of > 70% determined by High Pressure Liquid Chromatography. These peptides were selected as they represent in-vivo identified B-cell specific epitopes of conserved regions within C. pecorum MOMP20, known to be present in this population. The antigen was combined with a three-component adjuvant, containing Poly I:C (250 µg), Host Defence Peptide-Innate Defence Regulator IDR-1002 (500 µg), and Polyphosphazene EP3 (250 µg) (VIDO-Intervac, University of Saskatchewan, Canada). Vaccine preparation involved mixing the Poly I:C and IDR-1002 and incubating for 15 min at room temperature, with gentle rocking. Next, EP3 and the four peptides were added, with the final mixture incubated for a further 15 min at room temperature, with gentle rocking, then stored at 4C until use.20. Vaccination and placebo injections (Hartmann’s solution) were administered subcutaneously (0.5ml) into the interscapular region at first capture and then again at six months.
Urogenital and Ocular Swab DNA extraction and qPCR
DNA was extracted from ocular and urogenital swabs using the MagMAX™
CORE Nucleic Acid Purification Kit #A32702. A sterile swab was included as a control to monitor for contamination during the extraction process. A multiplex qPCR assay was used to quantify the following genes from each extracted swab sample, P. cinereus beta actin (HEX), 23S Chlamydia (ROX) and C. pecorum ompB (FAM); primers adapted from Hulse et al., 201836. PCR reactions were made to a final volume of 20 µl consisting of 10 µl of SensiFAST™ Probe, 400 nM of each primer, 200 nM of each probe, 4.4 µl of dH2O and 2 µl of DNA. The cycling conditions included initial denaturation for two minutes at 98°C, followed by 40 cycles of denaturation for 15 seconds at 98°C and a combined annealing and extension step for 30 seconds at 58°C. Samples were determined to be C. pecorum positive if the CT value of either 23S Chlamydia and/or C. pecorum ompB was ≤ 34.
Plasma anti-Major Outer Membrane Protein (MOMP) IgG ELISA
All concentrations of reagents, blocking and incubation conditions were determined by optimisation experiments using chequerboard titrations. In the optimised assay, 96 well flat bottom plates (Grenier #0030125150) were coated with 2 µg of recombinant MOMP serovar G (produced utilising methods previously described)9 in carbonate-bicarbonate buffer (Sigma-Aldrich #C3041) at 100 µl/well at 4 °C overnight. Wells were emptied and then blocked with 300 µl of 5% skim milk powder in PBS with Tween (0.05%) (PBST) at 37 °C for 1 hour. Wells were emptied and koala plasma was applied to the wells at a concentration of 1:400 at 100 µl/well and incubated at 37 °C for 1 hour. Wells were then emptied and washed five times with PBST with a microplate washer (Biorad model 1575). An in-house sheep anti-koala-IgG antibody13 100 µl was added to each well at a dilution of 1:8000 and incubated at 37 °C for 1 hour. Following incubation, wells were washed as described above. 100 µl of rabbit anti-sheep IgG HRP conjugated (Abcam #ab6747) was added to each well (1:20000 dilution) and incubated at 37 °C for 1 hour. Following incubation, wells were washed as described above, using PBS only. Finally, 100 µl of 3,3’, 5, 5’ Tetramethylbenzidine (Sigma-Aldrich #T5525) was added to each well and allowed to develop at room temperature, in the dark, for 20 minutes before 100 µl of 1M H2SO4 was added to each well. Optical density (OD) was read at 450 nm. Each plate contained a blank well (no antigen or plasma), the same negative control from a captive, C. pecorum negative koala and a standard curve based on doubling dilutions of a known strong positive sample, selected during optimisation, from a C. pecorum positive koala. The inter-assay coefficient of variation was < 15% and the intra-assay coefficient of variation was accepted if it was < 10%. Each sample was run in a well coated with antigen and one without antigen. All samples were run in duplicate, and the mean was calculated from these replicates. All OD values were blank adjusted; the OD value of the no-antigen well was subtracted from the OD value of the antigen and plasma well for each sample. The highest concentration standard was given a nominal value of 32. The other values were calculated relative to that standard, based on dilution. The OD values of the samples were compared to the standard curve using a 4-parameter logistic curve.
Lymphocyte stimulations
Three millilitres RPMI medium (Sigma-Aldrich #R7388), was incubated for 30 minutes at 37 °C. The same volume of heparanised blood from each koala was centrifuged at 3000 g for five minutes and the leukocyte fraction (buffy coat) was aspirated and suspended in the pre-incubated media. In duplicate, 5 µg of rMOMP genotype G in 25 µl of foetal calf serum (FCS) or, in the case of negative controls FCS 25 µl, was added to 220 µl of the buffy coat solution. Suspensions were then incubated for 12 hours at 37 °C then 750 µl of RNA later (Sigma-Aldrich #R0901) was added to each sample and the sample stored at room temperature for 24 hours, then frozen at -20 °C until processing.
RNA extraction and cDNA synthesis
RNA was extracted from the leukocyte suspensions using the RiboPure – Blood kit (Invitrogen #AM1928) according to the manufacturer’s protocol. Extracts were treated with RNAse-free DNAse, (Thermofisher #EN0521) and cDNA synthesis was then performed using the RevertAid First Strand cDNA Synthesis Kit (K1622) according to the manufacturer’s protocol.
IFNγ and IL-17A expression
A qPCR assay was used to estimate expression of the following genes: GAPDH, IFNγ and IL-17A37,38. Reactions were made to a final volume of 20 µl consisting of 10 µl of SsoAdvancedtm Universal SYBRR Green Supermix (BioRad), 0.5 µM of each primer with 6 µl of H2O (GAPDH) and 0.3 µM of primer with 6.8 µl of H2O (IFNγ and IL-17A) and 2 µl of DNA. Cycling conditions for the different genes were applied as previous37,38. All samples were run in duplicate with negative controls at each step. IFNγ and IL-17A expression for each koala for each time point were normalised to GAPDH by the 2−ΔΔCT method, where ΔΔCT = (CT of target − CT of GAPDH) − (CT of target − CT of GAPDH) and presented as a fold change in relation to the unstimulated sample.
Statistical analysis
All data analyses were performed using the R Statistical Environment (Version 3.6.1)39. Data were initially assessed using the ‘ggplot’ and ‘ggdensity’ functions from the ‘ggplot2’40 and ‘ggpubr’41 packages respectively. Shapiro-Wilk tests were used to check for normality. Data that did not conform to normality were log transformed.
Spearman’s Rank Correlations were used to test for relationships between immunological markers, chlamydial shedding, and disease variables. A variable was removed if a pair of variables had a correlation coefficient > 0.5 (or <–0.5)42. To normalise C. pecorum CT against swab yield (koala beta-actin DNA) from the same swab sample, ΔCT values were generated (ΔCT = CT of C. pecorum CT of beta-actin). Chlamydial shedding is expressed as the inverse of C. pecorum ΔCT values because, as chlamydial shedding decreases, C. pecorum ΔCT values increase.
The effect of treatment (vaccination or placebo), time since vaccination, their interaction and sex, was tested with different Linear Mixed Effects Models (LMM), constructed using the ‘lme4’ package43, with individual koala as the random factor. Dependant variables examined included clinical disease variables, specifically ocular disease scores, wet bottom score and paraovarian cyst score (females only), chlamydial shedding (ocular and urogenital ΔCT values), anti-MOMP plasma IgG values (log transformed) and cytokine expression (log transformed fold change).
Data availability
The data that support the findings of this study is available from the corresponding author upon request. There are no restrictions on data availability. This study is reported in accordance with ARRIVE guidelines.