Abstract

Chlamydia pecorum in koalas (Phascolarctos cinereus) is considered a sexually transmitted infection. Analysis of samples from koala joeys (<1 yr) suggested that mother-to-young direct transmission was also occurring. Further, evidence suggested that joeys from vaccinated mothers were less likely to contract infections than joeys with unvaccinated mothers.

Combating disease is a vital component to koala (Phascolarctos cinereus) conservation (Rhodes et al. 2011). Chlamydia pecorum poses the greatest threat because of its prevalence in populations and aggressive pathology (Polkinghorne et al. 2013). Diseases associated with C. pecorum in koalas include cystitis, scarring of the urogenital tract causing infertility, and proliferative conjunctivitis, which may lead to blindness (Polkinghorne et al. 2013). Sexual transmission has been implicated as the main route of infection in the koala. However, we have recently observed C. pecorum-like clinical signs in sexually inactive juvenile koalas, and vertical transmission may occur from mother to joey (Jackson et al. 1999). Perinatal transmission may be involved in some chlamydial species (Schachter et al. 1986). First, our study investigated whether C. pecorum infections are present in sexually inactive joeys. Second, we examined whether a recently developed koala antichlamydial vaccine may provide some protection against C. pecorum for koala joeys when their mothers were vaccinated.

To determine if C. pecorum infections were present in joeys, veterinarians provided samples from 11 sexually inactive joeys brought to Australia Zoo Wildlife Hospital (AZWH), Queensland, from April 2013 through December 2014 as orphans (e.g., mothers were victims of dog attack or motor vehicle collision). To determine if maternal immunization provided any protection to joeys, five mother–joey pairs were also sampled as part of a larger project (see Waugh et al. 2016 for the full methods involved in sampling the mother–joey pairs). The five koala pairs were captured at six monthly intervals, until the joeys were independent. Three mother koalas received the antichlamydial vaccine via the subcutaneous route as per Waugh et al. (2016), whereas two did not. Joeys from the mother–joey pairs were sampled twice: first, while the joey was dependent on the mother and sexually inactive, and again once the joey was >1 yr old, independent of the mother, and was assumed to be sexually active.

To determine infection status from each animal, urogenital (UGT) and ocular swabs were taken using cotton-tipped swabs (Copan, Interpath Services, Melbourne, Australia) by experienced wildlife veterinarians and stored at −20 C. We extracted DNA from the swabs followed by quantitative PCR (Waugh et al. 2016). The Clearview enzyme immunoassay test was also performed to detect the presence of chlamydial antigen against Chlamydiaceae-family-specific lipopolysaccharide as per Hanger et al. (2013). Disease status was determined by thorough veterinary examinations (Waugh et al. 2016). Animal use was conducted under the University of the Sunshine Coast Animal Ethics Committee permit number ANA1380.

Chlamydia pecorum DNA was detected by PCR in 27% (3/11) of koala joeys, which were positive for C. pecorum at one anatomical site. This indicated that young, mother-dependent joeys may have been infected directly from their mothers. Although the joeys had limited interactions with each other while in the care of AZWH, they shared the same animal handler. As a result, nonsexual transmission between joeys may also have been possible.

Of the five mother–joey pairs that were followed over time to determine the effect that maternal immunization might have on joeys, only joeys from unvaccinated mothers (2/2) developed C. pecorum infection, which occurred after they were independent from their mothers and were assumed to be sexually active (Table 1). By comparison, C. pecorum infections were not detected in any of the joeys from three vaccinated mothers (Table 1). This was evidence that an antichlamydial vaccination may have provided joeys with some level of protection against C. pecorum via maternal immunization. These data must be interpreted with caution because of the low sample size and follow-up studies need to be conducted. However, these positive data warrant further investigation.

Table 1

Chlamydia pecorum infection (quantitative PCR) and disease (clinical signs) status of koala (Phascolarctos cinereus) joeys at two times: first, while they were dependent on their mothers, and then when they were independent of their mothers. Joeys either had a vaccinated mother or an unvaccinated mother. Vaccines consisted of C. pecorum major outer membrane protein proteins adjuvanted with an immune-stimulated complex. Koalas were sampled between April 2013 and October 2014.

Chlamydia pecorum infection (quantitative PCR) and disease (clinical signs) status of koala (Phascolarctos cinereus) joeys at two times: first, while they were dependent on their mothers, and then when they were independent of their mothers. Joeys either had a vaccinated mother or an unvaccinated mother. Vaccines consisted of C. pecorum major outer membrane protein proteins adjuvanted with an immune-stimulated complex. Koalas were sampled between April 2013 and October 2014.
Chlamydia pecorum infection (quantitative PCR) and disease (clinical signs) status of koala (Phascolarctos cinereus) joeys at two times: first, while they were dependent on their mothers, and then when they were independent of their mothers. Joeys either had a vaccinated mother or an unvaccinated mother. Vaccines consisted of C. pecorum major outer membrane protein proteins adjuvanted with an immune-stimulated complex. Koalas were sampled between April 2013 and October 2014.

We have not delineated the route of transmission of C. pecorum between koala mothers and their joeys; there are several possibilities. In human studies, sexually transmitted diseases such as Chlamydia trachomatis have been transmitted to infants through exposure to the infected birth canal (Moodley and Sturm 2000). Another potential route of C. pecorum transmission is the fecal–oral route. Because koala joeys engage in pap feeding, it may be possible for C. pecorum to colonize the digestive tract of the joey before passing into the UGT region where it can cause disease (Rank and Yeruva 2014). Additionally, koala joeys spend the first 6 mo of their life in the pouch, which is in proximity to their mother's UGT and anus, resulting in frequent exposure to exudates, feces, and urine.

Newborns of some mammalian species rely on antibodies present within mother's milk as their primary source of protection against pathogens (Barrington and Parish 2001). The absence of infections in sexually active recently independent young from vaccinated mothers may be due to an increased presence of antichlamydial antibodies in the mothers' milk. The vaccine formulation used in this study is known to significantly increase the chlamydial specific immunoglobulin G response in the plasma and mucosa. These antibodies are also found to have significant in vitro Chlamydia neutralization potential (Waugh et al. 2015). The vaccine provides significant protection as well as a therapeutic effect in adult vaccinated koalas (Waugh et al. 2016), and antibodies passed on to the joey would explain a positive effect in joeys from vaccinating their mothers. Maternal immunization has been used as a strategy to decrease susceptibility of newborn infants to infections and works via transfer of vaccine-induced immunoglobulin G across the placenta (Lindsey et al. 2013). A targeted study on the effect of vaccination in mother–joey pairs is warranted, and further research into this effect in nonplacental mammals (such as the koala) is needed.

Our study was financially supported by the University of the Sunshine Coast Faculty of Science, Health, Education and Engineering Undergraduate Research Internship Scheme 2014. We were significantly supported by the Queensland Government (Department of Transport and Main Roads) and specifically by the Moreton Bay Rail project team. We thank the many groups that have supported the overall koala chlamydia work, including: Queensland Department of Environment and Heritage Protection, Moreton Bay Regional Council, Friends of Koala, Lismore, Koala Action Inc., Endeavour Veterinary Ecology, AZWH, Lone Pine Koala Sanctuary, Redland City Council, and Vaccine and Infectious Disease Organization, Canada. Specially, we thank the dedicated staff at Endeavour Veterinary Ecology for their help in capturing, radiocollaring, and tracking the koalas, and staff at AZWH for undertaking the health assessments and collecting samples from orphaned joeys. C.W. is supported by a postdoctoral fellowship at the Norwegian University of Science and Technology.

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