Diversity and Distribution of Escherichia coli in Three Species of Free-Ranging Australian Pinniped Pups

Anthropogenic activities and pollution are impacting marine environments globally. As a consequence, increasing numbers of human-associated phylotypes of Escherichia coli, an indicator of fecal contamination, have been found in both aquatic environments and marine mammals considered sentinels for marine health. The objective of this study was to determine the presence and diversity of E. coli in pups of three species of free-ranging pinnipeds in Australia. Fecal samples (n = 963) were collected between 2016 and 2019 from Australian sea lion (Neophoca cinerea), Australian fur seal (Arctocephalus pusillus doriferus) and long-nosed fur seal (Arctocephalus forsteri) pups from eight breeding colonies extending along the Southern Australian coast. E. coli were isolated from 842 (87.3%) samples and molecular screening was applied to assign isolates to E. coli phylotypes and sub-types. The human associated E. coli phylotype B2 was the most frequently isolated in all species at seven of the eight colonies, with 73.7% of all E. coli isolates belonging to this phylotype. Phylotype distribution did not differ significantly within or across species, breeding colonies or breeding seasons. Analysis of B2 isolates into sub-types showed a significant difference in sub-type distribution across breeding seasons at two colonies (Seal Rocks and Cape Gantheaume). The predominance of the B2 phylotype could indicate that all colonies are exposed to similar levels of anthropogenic pollution. This widespread occurrence of the human-associated E. coli phylotypes highlights the imperative for ongoing monitoring and surveillance of microbes in both the marine environment and sentinel species.


INTRODUCTION
The contamination of the marine environment with atypical microorganisms as a result of anthropogenic pollution poses unknown risks to both marine wildlife and the wider ecosystem (Oates et al., 2012;Baily et al., 2015). Fecal coliforms such as Escherichia coli are used globally as indicators of anthropogenic fecal contamination and as a measure of pollution of waterways and coastal ecosystems (Beversdorf et al., 2007;Schaefer et al., 2011;Ahmed et al., 2016). E. coli is a commensal species colonizing the intestinal tract of birds and mammals (Gordon and Cowling, 2003;Guenther et al., 2011). However, some strains of E. coli have virulence traits that render them opportunistic pathogens that can cause a range of diseases in multiple host species, including urinary tract infections, neonatal meningitis and septicemia (Russo and Johnson, 2000;Mora et al., 2009;Clermont et al., 2011). There are eight E. coli lineages (A, B1, B2, C, D, E, F, clade I), termed phylotypes, that differ in ecological niches, life histories, host affinities and pathogenic abilities; phylotypes B2 and D are commonly isolated from and associated with humans (Gordon and Cowling, 2003;Gordon et al., 2008). Many clinically relevant strains of E. coli that have the potential to cause extraintestinal disease in humans, domestic animals and birds belong to phylotype B2 (Johnson and Russo, 2002;Clermont et al., 2014).
Marine mammals are considered sentinels of ocean health with marine mammal health status providing insights into the health of the wider marine ecosystem (Bossart, 2010). Escherichia coli has been isolated from a number of aquatic mammal species including pinnipeds (Stoddard et al., 2008(Stoddard et al., , 2009Wallace et al., 2013;Delport et al., 2015;Power et al., 2016;Fulham et al., 2018;Mora et al., 2018), cetaceans (Schaefer et al., 2011;Melendez et al., 2019) and river otters (Oliveira et al., 2018). Despite this, little is known about the host and environmental factors that influence the diversity and prevalence of E. coli in marine mammals. Furthermore, the intestinal population of E. coli in mammals differs between individuals of the same species (Guenther et al., 2011). As a result, studies that focus on one species at a single time point are unlikely to accurately represent the diversity of E. coli within a species (Schierack et al., 2008(Schierack et al., , 2009Leser and Mølbak, 2009).
In the Southern hemisphere, E. coli has been isolated from multiple pinniped species including southern elephant seals (Mirounga leonina), Weddell seals (Leptonychotes weddellii), Antarctic fur seals (Arctocephalus gazella) and Australian sea lions (Neophoca cinerea) (Hernandez et al., 2007;Delport et al., 2015;Power et al., 2016;Fulham et al., 2018;Mora et al., 2018). These studies have characterized E. coli isolated from free-ranging individuals with a majority of samples collected from adults and a predominance of phylotype B2 identified; the prevalence of the B2 phylotype ranging from 49% (Fulham et al., 2018) to 65% . Comparisons of E. coli prevalence and phylotype distribution in captive and freeranging adult N. cinerea (Delport et al., 2015) determined a higher prevalence in captive individuals; however, in both captive and free-ranging animals, the human-associated phylotype B2 was the most frequently identified (Delport et al., 2015). A higher E. coli prevalence and differing trends in phylotype distribution was described in free-ranging N. cinerea pups from two colonies in South Australia (Fulham et al., 2018). Of the two colonies, phylotype B2 was more prevalent in samples from the colony closer to anthropogenic sources of pollution than the colony located further from human habitation (Fulham et al., 2018). It is unknown whether host or environmental factors could be contributing to the differences in phylotype distribution observed in E. coli isolated from free-ranging N. cinerea pups. For this reason, further investigations are required to understand the distribution and diversity of E. coli in marine mammal species in Australian waters, including other pinniped species such as the Australian fur seal (Arctocephalus pusillus doriferus) and long-nosed fur seal (Arctocephalus forsteri) where the prevalence and diversity of E. coli has not been reported previously.
The three pinniped species, N. cinerea, A. p. doriferus, and A. forsteri, inhabit numerous offshore colonies along the Australian coast from Western Australia to Tasmania (Kirkwood and Goldsworthy, 2013). All three species were subjected to commercial sealing practices during the nineteenth and twentieth centuries which decimated their numbers (Ling, 1999). Population recovery of each species from historical harvesting has differed greatly, with numbers of A. p. doriferus and A. forsteri steadily increasing since the 1960s and 1970s (Shaughnessy et al., 2015;McIntosh et al., 2018). In contrast, N. cinerea have undergone continual decline; the species is listed as endangered on the IUCN Red list . The reasons underlying these differing rates of population recovery are likely complex and multi-factorial and include differing life histories, breeding strategies, geographical distribution, extent of fisheries interactions and disease (Goldsworthy et al., 2009;Shaughnessy et al., 2011;Marcus et al., 2015). The geographical range of these species frequently overlap, with some species breeding at the same sites. This geographical proximity provides an unparalleled opportunity for comparative investigations of E. coli to better understand the role of host and environmental factors on E. coli prevalence and diversity.
The objective of this study was to characterize the diversity of E. coli in three species of free-ranging pinniped pups in Southern Australia, establishing base line data for future comparative studies and to assess potential effects of anthropogenic impacts.

Study Sites
Fecal swabs (n = 963) were collected from eight pinniped breeding colonies across multiple breeding seasons between 2016 and 2019 (Figure 1 and Table 1). Of the 963 fecal swabs collected, a total of n = 401 fecal swabs were from N. cinerea pups, n = 400 from A. p. doriferus pups and n = 162 from A. forsteri pups. Samples collected from N. cinerea pups at Seal Bay in 2016 and Dangerous Reef 2017 were collected and analyzed as part of a previous study (Fulham et al., 2018). Samples were collected across two breeding seasons for all sites except N. cinerea at Dangerous Reef and A. p. doriferus at Deen Maar Island, Cape Bridgewater and The Skerries. The proximity to human settlements differs for each colony; Dangerous Reef and Olive Island are the most remote locations, located approximately 33 and 15 km offshore, respectively, while Seal Rocks and Deen Maar Island are located less than 10 km offshore (Figure 1).

Sample Collection
Pinniped pups, ranging in age from 3 to 6 weeks for A. forsteri and A. p. doriferus and 2 to 6 weeks for N. cinerea, were captured by hand as part of ongoing health investigations and restrained in canvas pup bags with breathing holes for the duration of capture and sample collection (Fulham et al., 2018). Pups were sampled when mothers were absent. Fecal samples were collected by inserting a sterile swab (Copan, Brescia, Italy) covered by a lubricated sheath directly into the rectum or by swabbing a fecal sample passed by the pup during capture and restraint. Fecal swabs were then sub-sampled into Sterile FecalSwab TM (Copan, Brescia, Italy). All swabs were refrigerated at 4 • C until culture, usually within 7 days of collection. All sampling methods for N. cinerea and A. forsteri were approved by the Animal Ethics Committee at the University of Sydney (Protocol Nos. 2014/726 and 2017/1260); sampling methods for A. p. doriferus

E. coli Culture, Isolation, and Preservation
Fecal swab media was inoculated onto Chromocult R coliform agar (Merck, Millipore, Australia) and cultured plates incubated at 37 • C for 24 h. The E. coli colonies were identified by morphological features -being round, and dark blueviolet. Single colonies were selected and sub-cultured onto Chromocult R coliform agar plates to obtain pure cultures. To preserve cultures, pure E. coli isolates were grown in Luria Bertani (LB) broth (5 mL) at 37 • C for 24 h and 500 µL of broth culture was combined with 500 µL 70% glycerol in 1.0 mL cryovials and samples were stored at −80 • C.

Extraction of DNA From Preserved Bacteria
DNA was extracted using a boil preparation method. Preserved bacteria (40 µL) was inoculated into LB broth (150 µL) and incubated at 37 • C for 24 h. Broth cultures were centrifuged for 5 min at 4000 rpm (Eppendorf 5810 R, rotor: A-4-62), the supernatant was removed and the bacterial pellet was resuspended in molecular grade water (40 µL). Samples were then heated for 5 min at 95 • C and centrifuged for 5 min at 4000 rpm (Eppendorf 5430 R, rotor: FA-45-24-11-HS). Lysates were stored in 1.5 mL microcentrifuge tubes at −30 • C until PCR analysis.

Phylotyping of E. coli Isolates and Phylotype-Specific PCRs
To assign E. coli isolates to a phylotype, lysates were analyzed using a quadruplex PCR protocol following the methodology of Clermont et al. (2013). Isolates were assigned to a phylotype based on the presence or absence of four genes: ChuA, yjaA, TspE4.C2, and arpA. Isolates identified as A/C and D/E were further analyzed using two phylotype-specific PCRs. To assign isolates to phylotypes A or C, a singleplex PCR was conducted using the primers trpAgpC.1 and trpAgpC.2 with internal control primers trpBA.f and trpBA.r (Clermont et al., 2013). To assign isolates to phylotypes D or E, a singleplex PCR was conducted using primers ArpAgpE.f and ArpAgpE.r with internal control primers trpBA.f and trpBA.r (Clermont et al., 2013). All phylotyping PCRs were performed using GoTaq R Green Master Mix (Promega, Madison, United States) and included controls representing each phylotype , and a negative control containing PCR water. All reactions described above were resolved using gel electrophoresis (2% agarose w/v) conducted at 100 V for 30 min in TBE (Tris, boric acid, ethylenediaminetetraacetic acid) with SYBR safe gel stain (Invitrogen, Mulgrave, Australia). Product sizes were approximated against a HyperLadderII 50 bp DNA marker (Bioline, Sydney, Australia).

E. coli Phylotype B2 Sub-Typing
Isolates classified as B2 were further analyzed using two multiplex PCR panels to assign isolates to one of nine sub-groups using primers described by Clermont et al. (2014). Minor modifications were made to the PCR protocol by altering the annealing temperatures for both panels. Samples were assigned to subgroups based on the presence of the following fragments: putP, pabB, trpA, trpA, polB, dinB, icd, aes (IX), and aes (X). The chuA gene was included as an internal control. Multiplex panel 1 tested for sub-groups II, III, VI, VII, and IX, and multiplex panel 2 tested for sub-groups I, VI, V, and X. The PCRs for panels 1 and 2 were performed using GoTaq R Green 2X (Promega, Madison, United States) with the addition of MgCl 2 to increase the concentration to 2.0 mM MgCl 2 . The following PCR conditions were used: initial denaturation at 94 • C for 4 min; 35 cycles at 94 • C for 5 s, 59 • C for 20 s (panel 1) or 58 • C for 20 s (Panel 2), 72 • C for 20 s, 72 • C for 5 min and held at 4 • C. Isolates were either assigned one of the nine sub-groups or considered unassigned. All reactions were resolved using gel electrophoresis using methods described in section "Phylotyping of E. coli Isolates and Phylotype-Specific PCRs".

Distribution of E. coli Phylotypes in Pinniped Pups
Phylotyping of E. coli isolates showed only minor differences in distribution across the three species (Figure 2). There was no significant difference in E. coli phylotypes across species (p = 0.055), sampling sites (p = 0.437) or breeding season at colonies sampled over multiple breeding seasons; Seal Bay (p = 0.272), Olive Island (p = 0.199), Seal Rocks (p = 0.880), and Cape Gantheaume (p = 0.076) (Figure 3). The B2 phylotype was most frequently isolated from all samples with 73.7% of all E. coli isolates identified as B2, followed by B1, D, F, A, E, and C.
In N. cinerea, A. forsteri, and A. p. doriferus the most frequently identified phylotype was B2, with 62.5, 86.4, and 69.5% of isolates assigned to this phylotype, respectively (Table 2 and Figure 3). There were no E. coli isolates from A. p. doriferus pups assigned to phylotype E. Isolates from A. forsteri pups were the most diverse with seven phylotypes identified and phylotype C was only found in E. coli isolates from A. forsteri pups at Olive Island.

Distribution of B2 Sub-Types in Pinniped Pups
Isolates from N. cinerea, A. forsteri, and A. p. doriferus pups that were assigned to the B2 phylotype were further analyzed and assigned to one of nine B2 sub-types. There was no significant difference in B2 sub-type distribution across sampling sites (p = 0.768) or species (p = 0.121) (Figure 4). At Seal Bay and Olive Island there was no significant difference in B2 sub-type distribution across breeding seasons (p = 0.483 and p = 0.098, respectively) ( Figure 5). There was a significant difference in sub-type distribution across breeding seasons at Seal Rocks (p = 0.046) and Cape Gantheaume (p < 0.001). At Seal Rocks there was a decrease in the frequency of sub-types II, IV, VI, VII, IX, and X between seasons, while the frequency of sub-type III increased (Figure 4). The significant difference seen at Cape Gantheaume was due to an increase in sub-types II, V, VI, and IX and a decrease in the frequency of sub-types III, IV, and VII.
Sub-type distribution differed slightly between species with sub-type I only detected in a single N. cinerea pup sampled at Olive Island; sub-type X was only detected in one A. p. doriferus pup sampled at Seal Rocks. The most frequently isolated sub-type across all species was VI, followed by III, V, II, VII, IV, IX, I, and X (Figure 4). A total of 23.6% of B2 isolates could not be assigned (UA) to a sub-type.

DISCUSSION
This study reports the prevalence and diversity of E. coli isolated from free-ranging N. cinerea, A. p. doriferus, and A. forsteri pups at eight breeding colonies in Australia, finding no significant difference in E. coli prevalence or the distribution of phylotypes across species, colonies or breeding seasons. This is the first investigation of the comparative presence and distribution of E. coli in free-ranging A. p. doriferus and A. forsteri pups. The prevalence of E. coli was similar across all three species and supports previous reports in Antarctic pinnipeds including Antarctic fur seals, Southern elephant seals and Weddell seals Mora et al., 2018), and captive adult N. cinerea (Delport et al., 2015). Samples that could not be assigned to a sub-type were considered "unassigned" (UA). All nine sub-groups were identified in at least one of the three species.
FIGURE 5 | Distribution of E. coli B2 sub-types for each breeding season in each species across the eight sampling sites. Samples that could not be assigned were classed as "unassigned" (UA). Sub-type I was only identified in N. cinerea and sub-group X was only detected in A. p. doriferus pups.
Previous studies have suggested that E. coli is uncommon in marine mammals (Johnson et al., 1998;Hernandez et al., 2007) with a higher prevalence associated with proximity to humans (Stoddard et al., 2008;Delport et al., 2015).
In this study the differences in proximity to humans at each breeding colony and the high prevalence of E. coli observed in N. cinerea, A. p. doriferus, and A. forsteri pups could potentially indicate that proximity to humans is not the only factor contributing to E. coli prevalence in marine mammals.
The B2 phylotype was most frequently identified from pups of all three species, with 73.7% of all E. coli isolates assigned to this phylotype. This is the highest frequency of the B2 phylotype identified in pinnipeds in Australia; in free-ranging N. cinerea, B2 made up 67% of all isolates in adults (Delport et al., 2015). It has been suggested that strains belonging to the B2 phylotype are well adapted to the intestinal environment of mammals (Gordon and Cowling, 2003;Nowrouzian et al., 2006), which could be a contributing factor to the high frequency identified in this study. The B2 phylotype was the most frequently isolated phylotype at seven of the eight breeding colonies during every breeding season sampled. It was previously hypothesized that the difference in phylotype distribution observed in freeranging N. cinerea pups was due to colony location (Fulham et al., 2018), however, the results from this study indicate that geographical location and proximity to humans, is not a contributing factor to E. coli diversity in N. cinerea, A. p. doriferus, or A. forsteri pups.
The prevalence of E. coli in free-ranging pinniped pups is likely diet related; E. coli is a lactose fermenter and as the sampled pups were feeding solely on milk, high E. coli prevalence is not unexpected (Fulham et al., 2018). In adult N. cinerea, E. coli prevalence from free-ranging individuals was significantly lower (7.7%) compared to captive individuals (84%) suggesting that E. coli does not occur naturally in freeranging adult N. cinerea (Delport et al., 2015). Marine wildlife species that forage in coastal or nearshore waters are at greater risk of exposure to pathogens in fecal bacteria as a result of anthropogenic pollution compared to species further removed from anthropogenic influence (Oates et al., 2012). In Antarctic pinnipeds, it was hypothesized that the presence of humanassociated E. coli could be due to the geographic mobility of the animals sampled (Mora et al., 2018). Free-ranging N. cinerea, A. p. doriferus, and A. forsteri adults occupy large geographical ranges (Shaughnessy et al., 2011(Shaughnessy et al., , 2015McIntosh et al., 2018), encountering a variety of environments and environmental conditions that could influence their exposure to anthropogenic pollution. In other pinniped species, there is evidence of maternal transmission of gut microbes (Nelson et al., 2013b), however, it is unknown whether the acquisition of E. coli in pinniped pups in this study is environmental or through maternal transfer. Understanding the prevalence of E. coli and phylotype diversity in free-ranging adults could provide valuable insights into the factors that contribute to the acquisition of E. coli and the trends in E. coli prevalence and phylotype distribution seen in pups.
Studies investigating the transfer of E. coli between humans and wildlife species are limited to terrestrial environments. Generally, E. coli isolated from wildlife species that share habitats and have higher levels of interactions with humans and livestock were genetically similar to E. coli isolated from humans (Goldberg et al., 2007;Rwego et al., 2008a,b). This genetic similarity suggests E. coli transmission between these species in shared habitats is likely a result of indirect contact through contaminated environmental sources rather than direct contact (Goldberg et al., 2007). For example, E. coli isolated from the feces of banded mongoose (Mungos mungo) was genetically similar to E. coli isolated from human fecal waste in their environment, highlighting the importance of indirect routes of transmission (Pesapane et al., 2013). These results suggest that the acquisition of human-associated bacteria by wildlife species is linked to fecal contamination of the environment.
Coastal environments can be contaminated by fecal pollution through a number of different sources including sewage and storm water runoff from agricultural, urban and commercial land (Crain et al., 2009;Pandey et al., 2014). The population size and density of people in coastal towns will influence the amount of bacterial contamination of the environment from runoff, with higher density resulting in higher levels of fecal bacteria (Pandey et al., 2014). The population size of towns closest to the pinniped breeding colonies sampled in this study varied, however, the prevalence of E. coli did not differ across colonies or seasons, suggesting that the contribution of human population density to trends in E. coli prevalence observed requires further investigation. Previous studies have determined that E. coli isolates from wildlife species occupying habitats in close association with humans or that are exposed to fecal pollution are more likely to belong to phylotype B2 than those isolated from wildlife living in isolation from humans (Gordon and Cowling, 2003). E. coli isolates belonging to B2 and D phylotypes have been found in treated sewage, suggesting a greater ability to survive treatment processes (Anastasi et al., 2010). The survival of these phylotypes in wastewater coupled with increasing pollution of coastal ecosystems presents a higher risk of transfer of human-associated bacteria into the marine environment. The predominance of the B2 phylotype across the three pinniped species studied could therefore suggest that all species are exposed to similar levels of anthropogenic pollution. This could be important for pup health, given that strains belonging to this phylotype are associated with extraintestinal disease in humans (Dale and Woodford, 2015).
Ocean currents and tides are also potential factors contributing to the high prevalence of E. coli in pinnipeds. The main source of ocean pollution is of terrestrial origin (Robinson et al., 2017) and as a consequence, coastal areas in close proximity to populated areas are exposed to higher levels of anthropogenic pollution (Partelow et al., 2015). The marine environment, being connected over longer timescales (Jönsson and Watson, 2016) has greater connectivity compared to terrestrial environments, with fewer physical barriers between areas. Consequently, the pathways of water that flow along coastlines can influence the dispersal of anthropogenic pollutants. Wastewater effluent is a known source of humanassociated E. coli phylotypes (de Stoppe et al., 2017) and E. coli that originate from wastewater have an enhanced capacity to survive in the marine environment, able to survive for several days outside of a host (Rozen and Belkin, 2001). In addition, E. coli can attach to particles in the water column, facilitating movement in the marine environment (Mallin et al., 2000). This enhanced survival and attachment to particles in the water column could result in prolonged environmental persistence of E. coli, facilitating acquisition by marine wildlife. The similarity in E. coli distribution across species and breeding colonies in this study could be indicative of the high degree of connectivity of marine ecosystems (Crain et al., 2009;Robinson et al., 2017) or similar anthropogenic pollution across this large geographical range.
The identification of B2 sub-types I, II and IX are of particular interest given that clonal complexes that belong to these subtypes are commonly associated with disease in avian species and humans (Clermont et al., 2014;Riley, 2014). ST131 and ST95 strains belonging to sub-types I and IX, respectively, are associated with disease caused by extraintestinal pathogenic E. coli (ExPEC) (Dale and Woodford, 2015). These subtypes or strains have previously been identified in Antarctic pinnipeds Mora et al., 2018). Assignment of E. coli isolates to strains was not undertaken in this study, however, given the presence of B2 sub-types associated with pathogenic strains, further analysis of B2 isolates will assist our understanding of the potential risks posed to pup health by their presence.
It is also important to consider that E. coli is a very small component of the intestinal microbiota in pinnipeds (Nelson et al., 2013a,b;Delport et al., 2016). However, the ease with which E. coli can be cultured and characterized has resulted in E. coli being commonly used for monitoring fecal contamination of marine environments (Beversdorf et al., 2007). For this reason, it can be a useful indicator of the diversity of E. coli phylotypes present in marine mammals, and a relatively high prevalence may suggest greater exposure to anthropogenic pollution. Sampling of substrate and water surrounding breeding colonies and comparing those E. coli phylotypes with diversity seen in pinniped pups could provide useful insights into potential sources of environmental contamination. Investigating the presence of specific markers through microbial source tracking could also be utilized to determine the origin of E. coli found in both wildlife species and contaminated environments.
The presence of bacteria and protozoa that are associated with humans should be explored to further understand potential pathogen transmission from anthropogenic sources into the marine environment. Toxoplasma gondii, Campylobacter spp. and Salmonella spp. have been isolated from marine mammals inhabiting coastal environments, including gray seals (Halichoerus grypus), Californian sea otters (Enhydra lutris nereis) and northern elephant seals (Stoddard et al., 2005;Oates et al., 2012;Shapiro et al., 2012;Baily et al., 2015). Campylobacter spp. isolated from H. grypus were genetically similar to isolates commonly found in agricultural and human sources, demonstrating the dissemination of a human pathogen into the marine environment (Baily et al., 2015). Similar investigations in Australian pinniped species could enable the identification of the source and dissemination of anthropogenic microbial pollution into the Australian marine environment.
In this study, the predominance of the human associated B2 phylotype and similarity of E. coli prevalence and phylotype diversity seen across species, colonies and breeding seasons could indicate that all colonies are exposed to similar levels of anthropogenic pollution. This widespread occurrence of human associated phylotypes highlights the need for ongoing monitoring and surveillance of microbes in both the marine environment and sentinel species, particularly those with potential pathogenicity for marine mammals. Future investigations should focus on whether E. coli is an atypical bacterium in these pinniped species and determine its reliability as an indicator of marine pollution.

DATA AVAILABILITY STATEMENT
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

ETHICS STATEMENT
The animal study was reviewed and approved by the Animal Ethics Committee at the University of Sydney and Phillip Island Nature Parks.

AUTHOR CONTRIBUTIONS
MF, RG, and MP contributed to conceptualization and design of the study. MF and RG collected samples from pinniped pups. MF completed laboratory analysis of samples and data analysis. MF drafted the manuscript. RG and MP participated in revising the manuscript. All authors contributed to the article and approved the submitted version.

FUNDING
Collection and analysis of samples was funded by the Ecological Society of Australia through the provision of the Holsworth Wildlife Research Endowment and the Sydney School of Veterinary Science at University of Sydney through the Late Dorothy Minchin Bequest. Field work support was provided by the Hermon Slade Foundation.