Chlamydia infection is known to impact the health of koalas (Phascolarctos cinereus) in New South Wales (NSW) and Queensland, but the clinical significance of Chlamydia infections in Victorian koalas is not well described. We examined the prevalence of Chlamydia infection and assessed associated health parameters in two Victorian koala populations known to be Chlamydia positive. The same testing regimen was applied to a third Victorian population in which Chlamydia had not been detected. We examined 288 koalas and collected samples from the urogenital sinus and conjunctival sacs. Detection and differentiation of Chlamydia species utilized real-time PCR and high-resolution melting curve analysis. Chlamydia pecorum was detected in two populations (prevalences: 25% and 41%, respectively) but only from urogenital sinus swabs. Chlamydia was not detected in the third population. Chlamydia pneumoniae was not detected. Chlamydia pecorum infection was positively associated with wet bottom (indicating chronic urinary tract disease) in one Chlamydia-positive population and with abnormal urogenital ultrasound findings in the other Chlamydia-positive population. The prevalence of wet bottom was similar in all populations (including the Chlamydia-free population), suggesting there is another significant cause (or causes) of wet bottom in Victorian koalas. Ocular disease was not observed. This is the largest study of Chlamydia infection in Victorian koalas, and the results suggest the potential for epidemiologic differences related to Chlamydia infections between Victorian koalas and koalas in Queensland and NSW and also between geographically distinct Victorian populations. Further studies to investigate the genotypes of C. pecorum present in Victorian koalas and to identify additional causes of wet bottom in koalas are indicated.

In koalas (Phascolarctos cinereus), Chlamydia infection may be subclinical or may be associated with a range of clinical diseases, including ocular and urogenital tract disease (Polkinghorne et al. 2013). Koalas with chronic urinary tract disease can have yellow-brown urine-staining wetness around the rump, commonly referred to as wet bottom or dirty tail. Factors that influence disease expression within a population are not fully understood but may include the species and strain of Chlamydia present, undescribed immunologic or other genetic differences between geographically divergent koala populations, and the presence of other stressors, such as coinfection with the potentially immunosuppressive virus, koala retrovirus (KoRV) and overcrowded habitat (Blanshard and Bodley 2008). Populations of koalas in Victoria, Queensland, and New South Wales (NSW), Australia, differ markedly in regard to KoRV prevalence, with 100% in Queensland and NSW and lower in Victorian populations (Tarlinton et al. 2005; Simmons et al. 2012), and population density (low in Queensland and NSW and high in many Victorian populations).

Studies have shown that Chlamydia pecorum and Chlamydia pneumoniae are prevalent in koala populations in Queensland and NSW (Polkinghorne et al. 2013), but no recent studies of Victorian koalas have been published. The historical studies of Chlamydia in Victorian koalas were limited by the use of less-sensitive diagnostic techniques than the DNA-based assays that are now available. These techniques did not differentiate Chlamydia species (McColl et al. 1984; Mitchell 1988; Martin and Handasyde 1990).

We used a DNA-based assay (real-time PCR-high-resolution melting curve analysis; [qPCR-HRM]) to detect and differentiate Chlamydia species in three free-ranging high-density populations of koalas in Victoria, Australia. We also conducted comprehensive physical examinations of each sampled koala to examine the relationship between Chlamydia infection and any manifestation of clinical disease.

Clinical examinations and sample collection

Clinical examinations and sample collection occurred during routine population management operations conducted by the Victorian Department of Sustainability and Environment (DSE) and Parks Victoria (PV). Three populations were studied, French Island (37°55′S, 147°44′E; 63 females and one animal of unrecorded sex examined; May and October 2010), Raymond Island (38°20′58″S, 145°23′5″E; 51 males and 53 females examined; June and October 2010), and Mt Eccles National Park (30°04′44″S, 141°53′19″E; 120 females examined; September and October 2010). All these locations are considered to contain an overabundance of koalas, thus necessitating population control measures. All animal handling and sampling was conducted under a permit issued by DSE and PV (permit 10005388). The University of Melbourne Animal Ethics Committee granted approval for physical examinations and sample collection on animals that were captured and restrained for these management operations (ethics 1011687.1).

Physical examinations were performed by using existing methodologies and scoring systems wherever possible. Body condition was scored from 1 (emaciated) to 5 (excellent) by palpation of the scapular spine and associated muscle mass. Estimation of age by tooth wear class was performed, as described previously (Martin 1981). Grading of ocular abnormalities was performed by using methods described by Griffith (2010). Each eye was graded as normal (0), mild (1), moderate (2), or severe (3) for the parameters of chemosis, proliferation of conjunctiva, amount of discharge, and erythema. Scores were then summed to give a total eye score. Any other ocular abnormalities were also noted. Ultrasound examinations were performed to evaluate the urogenital system, as described by Mathews et al. (1995) with modifications. Ultrasound examination assessed and measured the dimensions of the wall and lumen of both uteri (in females), the urinary bladder, and the sagittal length, horizontal width, and dorsal-ventral width of each kidney. Any other urogenital abnormalities, including paraovarian cysts, were also noted. Ultrasound examinations were only performed on animals that were anesthetized and did not have pouch young. Wet bottom was scored from 0 (absent) to 10 (severe), based on the system of Flanagan (2009). Abdominal palpation, including assessment of gut fill and hydration status, was performed, as described by Blanshard and Bodley (2008). Palpation of superficial inguinal, superficial axillary, and rostral mandibular lymph nodes were conducted. Physical examinations also included an examination of the pouch for the presence of young.

Swab samples were collected from each koala following clinical examination, as described by Blanshard and Bodley (2008). Sterile cotton swabs (Copan Italia, Brescia, Italy) were used to sample the conjunctivae of both eyes and the urogenital sinus (females) or penile urethra (males). Swabs were stored at room temperature for no more than 6 h and then stored at −20 C or −70 C.

The DNA extraction

Swab samples from both left and right eyes from each animal were combined for extraction, while swabs from the urogenital sinus or penile urethra were extracted separately. Sample material was resuspended in 500 µL of phosphate-buffered saline and the VX Universal Liquid Sample DNA Extraction Kit (Qiagen, Hilden, Germany) and a Corbett X-tractor Gene Robot (Qiagen) were used to extract DNA from 200 µL of each sample. Each 96-well plate for DNA extraction had at least six negative control wells containing sterile media only. The negative extraction wells were distributed across different rows and columns of the plate. Partially full plates had at least one negative extraction control well per column. All plates had one positive extraction control well containing stock material of Chlamydia felis, C. pecorum, or C. pneumoniae. Following extraction, eluted DNA (100 µL) was stored at −70 C until further analysis.

Chlamydia detection by qPCR

Real-time PCR was used to screen all swab samples for Chlamydia DNA. Each sample was tested in duplicate. The forward PCR primer (5′-GATGAGGCATGCAAGTC-3′) and the reverse PCR primer (5′-TTACCTGGTACGCTCAAAT-3′) targeted a conserved region of the 16SG rRNA gene, and all reactions were performed using an Mx3000P Quantitative PCR thermocycler (Agilent Technologies, Chatswood, NSW, Australia), using previously described conditions (Robertson et al. 2009, 2010). Immediately following completion of the PCR cycles, a melting curve was generated by increasing the temperature from 55 C to 95 C. The profile of the melting curve for each clinical sample was compared with curves generated from control samples of Chlamydia DNA. Samples were determined to be positive for Chlamydia DNA if the cycle threshold value was less than 35 and the profile of the curve was consistent with those generated from positive control samples. Positive qPCR controls consisted of the target sequence from clinical samples of C. pecorum or C. pneumoniae cloned in pGEM-T (Promega, Madison, Wisconsin, USA) according to the manufacturer's instructions. Negative qPCR controls used sterile water. The detection limit of the assay for each of C. pecorum and C. pneumoniae was determined by testing eight, ten-fold serial dilutions of 1×108 copies of the cloned product in triplicate.

Chlamydia identification by qPCR-HRM curve analysis

Samples that were positive by qPCR were retested for Chlamydia species identification by qPCR in triplicate using the same conditions, as previously mentioned, but with a RotorGene Thermocycler 6000 (Qiagen). Immediately following the completion of the PCR cycles, melting curves were generated by increasing the temperature from 78 C to 90 C at ramps of 0.3 C, as described (Robertson et al. 2009, 2010). The HRM curve analysis was performed by using the software Rotor-Gene 1.7.27 and the HRM algorithm provided, as described previously (Robertson et al. 2009, 2010), except normalization regions of 83.0–83.5 C and 85.0–85.5 C were applied. All qPCR-HRM curve analyses included positive controls of cloned and purified Chlamydia DNA from clinical samples of C. pecorum and C. pneumoniae. Positive controls were run in duplicate for each Chlamydia species for each qPCR-HRM curve analysis.

Statistical analysis

Statistical analyses were performed with the software program PASW Statistics 18 (SPSS, Chicago, Illinois, USA). Population differences in prevalence of C. pecorum, fecundity, prevalence of urinary tract disease, and body condition were examined using chi-square tests for independence. The associations between the independent variables (location, age, and C. pecorum status) and measures of health, including wet bottom, reduced gut fill, reduced hydration, peripheral lymphadenomegaly, poor body condition, abnormal urogenital ultrasound findings (in females), and absence of young (in females) were determined. Univariable analyses (chi-square tests for independence) were used to identify relationships between each independent variable and each health measure that had a significance level of P<0.25. These were then used in binary logistic regression multivariable models, and a backwards, stepwise elimination method was used with the least-significant variable removed after each step. The models were run for each individual population, for C. pecorum-positive populations (Raymond Island and Mt Eccles National Park) combined, and for all populations combined. Statistical significance was set at P<0.05.

Physical examination

The majority of the animals were found to be in good health, as assessed by body condition score, hydration status, gut fill status, and lymph node palpation (Table 1). By examination of tooth wear, the majority of animals were in the mature age class. Wet bottom and ultrasound abnormalities were detected in all three populations (Table 1). Ocular pathology, other than pathology consistent with trauma, was not observed.

Table 1.

Physical examination findings for koalas (Phascolarctos cinereus) in each of the study populations in Victoria, Australia, 2010.

Physical examination findings for koalas (Phascolarctos cinereus) in each of the study populations in Victoria, Australia, 2010.
Physical examination findings for koalas (Phascolarctos cinereus) in each of the study populations in Victoria, Australia, 2010.

Analyses by qPCR-HRM

Swab samples were tested for Chlamydia DNA using qPCR-HRM analyses. All positive control samples of C. pecorum and C. pneumoniae DNA included in the qPCR-HRM analyses produced melting curves expected of amplicons generated using the oligonucleotide set 16SG at a ramp of 0.3 C/s, as previously described (Robertson et al. 2009, 2010). Each qPCR-HRM curve analysis included a qPCR-negative control and DNA extraction negative control. No PCR products were amplified in these negative control reactions. The limit of detection for each of C. pecorum and C. pneumoniae was 10 copies per reaction.

Prevalence of Chlamydia infection

The prevalence of Chlamydia varied between the three koala populations studied (Table 2). Infection with C. pecorum was detected in two populations (Raymond Island and Mt Eccles National Park). In both these populations C. pecorum DNA was detected only in samples collected from the urogenital tract. Chlamydia DNA was not detected from ocular samples. No swab samples were positive for C. pneumoniae. Chlamydia DNA was not detected from any of the 63 animals from French Island, suggesting this population may be free from Chlamydia infection. The Raymond Island population had a significantly higher prevalence of urogenital infection with C. pecorum than the population at Mt Eccles National Park (41.3% and 25%, respectively, P = 0.01). At French Island and Mt Eccles National Park, only female animals were sampled, while at Raymond Island approximately equal numbers of both sexes were sampled. There was no significant difference in the prevalence of urogenital tract C. pecorum infection between males and females on Raymond Island (45% and 37%, respectively, P = 0.53).

Table 2.

Prevalence of Chlamydia pecorum in male and female koalas (Phascolarctos cinereus) in each of the study populations in Victoria, Australia, 2010, as determined by real-time PCR and high-resolution melting curve analysis.

Prevalence of Chlamydia pecorum in male and female koalas (Phascolarctos cinereus) in each of the study populations in Victoria, Australia, 2010, as determined by real-time PCR and high-resolution melting curve analysis.
Prevalence of Chlamydia pecorum in male and female koalas (Phascolarctos cinereus) in each of the study populations in Victoria, Australia, 2010, as determined by real-time PCR and high-resolution melting curve analysis.

Associations between Chlamydia infection and disease

Wet bottom and abnormal urogenital ultrasound findings

The presence of wet bottom was used as an indicator of urinary tract disease, and the presence of structural abnormalities on ultrasound examination was used to indicate urogenital tract disease. The prevalence of wet bottom was similar among all three populations (Table 1). Mt Eccles National Park had a significantly higher percentage of animals with abnormal urogenital ultrasound findings when compared with French Island (P = 0.04). There was no significant difference between the prevalences of abnormal urogenital ultrasound findings on Raymond Island and French Island.

The relationships between clinical signs of disease and Chlamydia infection for the two Chlamydia-positive populations are shown in Table 3. Data from the C. pecorum-positive populations (Raymond Island and Mt Eccles National Park) were combined, and using Chlamydia status as the independent variable, the likelihood of having wet bottom or abnormal ultrasound findings were each estimated using binary logistic regression. We found significant association between urogenital C. pecorum infection and presence of wet bottom (odds ratio [OR] = 2.16, 95% confidence interval [CI] = 1.22, 3.82, P = 0.008). There was also a significant association between urogenital C. pecorum infection and abnormal ultrasound findings (OR = 3.18, 95% CI = 1.08, 9.38, P = 0.036). Only females were included in the analysis of ultrasound findings. No other significant associations were observed.

Table 3.

Relationship between Chlamydia pecorum infection status and clinical disease (wet bottom and ultrasound abnormalitiesa) in koalas (Phascolarctos cinereus) at Raymond Island and Mt Eccles National Park, Victoria, Australia, 2010.

Relationship between Chlamydia pecorum infection status and clinical disease (wet bottom and ultrasound abnormalitiesa) in koalas (Phascolarctos cinereus) at Raymond Island and Mt Eccles National Park, Victoria, Australia, 2010.
Relationship between Chlamydia pecorum infection status and clinical disease (wet bottom and ultrasound abnormalitiesa) in koalas (Phascolarctos cinereus) at Raymond Island and Mt Eccles National Park, Victoria, Australia, 2010.

When animals from Raymond Island and Mt Eccles National Park were analyzed as separate populations, we found a significant association between the presence of wet bottom and C. pecorum infection for Raymond Island koalas (OR = 2.78, 95% CI = 1.24, 6.24, P = 0.013) but not for koalas at Mt Eccles National Park (OR = 1.59, 95% CI = 0.69, 3.36, P = 0.28). The two ORs were not significantly different (P = 0.34). In addition, there was a significant association between abnormal urogenital ultrasound findings and C. pecorum infection at Mt Eccles National Park (OR = 4.18, 95% CI = 1.09, 16.04, P = 0.037) but not at Raymond Island (OR = 2.75, 95% CI = 0.28, 26.61, P = 0.38). The two ORs were not significantly different (P =  0.76). Only females were included in this analysis of ultrasound findings.

Fecundity

Fecundity rates (measured by the presence of pouch and back young during September and October) were inversely proportional to Chlamydia infection rates, with the lowest fecundity rate recorded on Raymond Island (14%), an intermediate rate recorded at Mt Eccles National Park (39%), and the highest rate on French Island (44%). However, when the association between Chlamydia status and fecundity was examined for individual animals, no significant associations were found.

Age, poor body condition, and other measures of health

Although C. pecorum infection rates increased with age class in Chlamydia-positive populations (24% in sexually immature animals, 32.7% in mature animals, and 54% in aged animals in the Raymond Island and Mt Eccles National Park populations, combined), no statistical significant association between Chlamydia infection rates and age class was found using chi-square tests for independence. No significant associations were found between the measured health parameters (abnormal health status, enlarged peripheral lymph nodes, poor body condition, reduced gut fill, and reduced hydration) and the independent variables of location, age, and C. pecorum infection status when populations were analyzed individually or combined.

We systematically assessed the epidemiology and clinical significance of Chlamydia infection in a large number of Victorian koalas. The large sample size allowed relatively precise estimates of association.

We did not detect C. pneumoniae in any koalas and detected C. pecorum only in urogenital swabs. Chlamydia was not detected in ocular swabs, and ocular disease was not observed. In koala populations in Queensland and NSW, C. pecorum and C. pneumoniae have been detected at ocular and urogenital sites and have been associated with ocular and urogenital disease (Jackson et al. 1999; Polkinghorne et al. 2013). In Queensland and NSW populations, the prevalence of C. pneumoniae is typically lower than that of C. pecorum, and the levels of the organism present during infection are typically lower also (Jackson et al. 1999; Polkinghorne et al. 2013). Although C. pneumoniae appears to have a lower pathogenic potential than C. pecorum (Jackson et al. 1999), it is possible that a synergistic relationship between the two species could contribute to disease expression (Schiller et al. 1997; Dean et al. 2008). The absence of detectable C. pneumoniae in this study could, therefore, help to explain the absence of ocular disease and the apparently lower severity of wet bottom in these animals (wet bottom scores of 1–2) compared with the very severe disease observed in some populations of NSW and Queensland koalas (Blanshard and Bodley 2008; Polkinghorne et al. 2013). It is also possible that the genotype of C. pecorum in the Victorian populations is different from that in the NSW and Queensland populations and that this contributes to differences in tissue tropism and disease expression (Higgins et al. 2012).

The presence of other stressors should also be considered. The koala populations in this study were generally in good health, and infection rates with KoRV (a suspected cause of immunosuppression) are lower in Victorian koalas compared with koalas in Queensland and NSW. Previous investigators have recorded KoRV infection rates of 34.5% at Raymond Island and 21% at French Island, compared with 100% KoRV infection in koalas in Queensland and NSW (Simmons et al. 2012). All these factors may influence Chlamydia infection rates and disease expression.

The prevalence of C. pecorum infection differed between the Chlamydia-positive populations (25% at Mt Eccles National Park and 41% at Raymond Island). Variation in the prevalence of C. pecorum infection (8–87%) has also been observed in koala populations in Queensland and NSW (Polkinghorne et al. 2013). The higher prevalence of C. pecorum infection on Raymond Island might be related to higher stress experienced by this population. At Raymond Island, koalas exist in a profoundly disturbed, small, island habitat that has been highly developed. The stress on their preferred browsing trees, the coastal manna gum (Eucalyptus viminalis), is evident on examination of the canopy. Although the koalas within Mt Eccles National Park are overabundant, they exist in a much larger area, with stands of browsing trees in reasonable health, and the area is much less disturbed by human encroachment.

The prevalence of wet bottom was not significantly different among the three populations, but there was a higher prevalence of ultrasound abnormalities in the population of koalas at Mt Eccles National Park. It is possible that some animals with wet bottom were infected with undetectable levels of C. pecorum; however, the high prevalence of wet bottom on French Island (a seemingly Chlamydia-free population) suggests that there is another cause (or other causes) of wet bottom in Victorian koalas. The wet bottom scoring system (Flanagan 2009) might actually describe clinical observations arising from multiple etiologies with overlapping severities. Higher wet bottom scores could be due to Chlamydia infection, while lower scores (such as those observed in this study) may be due to other causes. Investigation of the cause of wet bottom in the koala population on French Island would be a useful starting point to examine this hypothesis.

Similar to the uncertainty surrounding the relationship between C. pecorum and wet bottom in these animals, some of the animals with abnormal ultrasound findings that were negative for Chlamydia infection may have had an undetectable level of infection or a Chlamydia infection that had resolved, leaving chronic genital tract changes (Hemsley and Canfield 1997), or there may be other causes for the abnormalities. Variation in the prevalence of abnormal ultrasound findings among populations could also be explained by the influence of the C. pecorum genotype and the presence of other stressors, as outlined previously.

The Raymond Island population showed no significant sex bias in the prevalence of urogenital infection with C. pecorum. This is consistent with a study in Queensland koalas that also used molecular detection techniques (Jackson et al. 1999). Earlier studies that assessed clinical signs or postmortem pathologic changes consistent with Chlamydia-induced disease found a higher prevalence of disease in female koalas than males (Obendorf 1983). Although the prevalence of C. pecorum infection increased with age class, we found no statistically significant association between Chlamydia infection rate and age class. This supports findings from Queensland koala populations suggesting that horizontal transmission from females to young is important (Jackson et al. 1999).

Fecundity differed between populations. Only animals sampled in spring (September and October) were included in this analysis to control for the effect of season. The percentage of females with young is at its lowest in spring; however, this was when the majority of the animals were sampled for this study. The decrease in fecundity corresponded to increasing infection rates with C. pecorum in the three populations; however, we detected no significant association between C. pecorum infection and the absence of young. Other factors that may contribute to these differences should be considered.

When considering the management of Victorian koalas, it is important to note the variation in C. pecorum prevalence in the context of translocation of animals between populations or movement of animals into captivity. Future studies to ascertain the genotypes of C. pecorum present, and if they vary among geographic locations and in disease associations, would also be helpful for informing translocation practices. Such studies, combined with investigations of other possible causes of wet bottom and the influence of KoRV on Chlamydia infection and disease expression, would contribute to our understanding of koala health and disease.

We gratefully acknowledge Helen McCracken, Kate Bodley, Jenny Kingston, Kath Handasyde, Carol Hartley, Hayley Blacker, Rebecca Agnew, Paola Vaz, Mauricio Coppo, Glenn Browning, Damien Higgins, Jon Hanger, Peter Timms, Charles Wang, and DPI and PV staff for help and advice. This study was supported by the Department of Environment and Resource Management, Queensland, and by the Sacramento Zoo, California.

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