ABSTRACT

The invasive ectoparasite Sarcoptes scabiei affects the welfare and conservation of Australian marsupials. Molecular data suggest that spillover from other hosts may be responsible for the emergence of this infectious disease, but the scale of such studies is limited. We performed expanded molecular typing of the S. scabiei mitochondrial cox1 gene from 81 skin scrapings from infested wombats (Vombatus ursinus), koalas (Phascolarctos cinereus), red foxes (Vulpes vulpes), and dogs (Canis lupus familiaris) across Australia. Combined with existing S. scabiei sequences, our analysis revealed 16 haplotypes among Australian animals, sharing between 93.3% and 99.7% sequence similarity. While some sequences were unique to specific hosts or to Australia, key haplotypes could be detected across several marsupial hosts as well as to wild or domestic canids in Australia. We identified 43 cox1 haplotypes with many Australian haplotypes identical to S. scabiei mites from inside and outside Europe. We concluded that multiple introduction events were plausible explanations to the origin and emergence of this parasite into Australian marsupials and that disease spillover from canids was likely. Together, our greatly expanded S. scabiei sequence dataset provided a more nuanced picture of both spillover and sustained intraspecific transmission for this important parasite.

Sarcoptes scabiei is an emerging invasive ectoparasite known to infest more than 100 mammal species globally, including humans (Pence and Ueckermann 2002; Tompkins et al. 2015). Signs of sarcoptic mange (scabies in human) include severe dermatitis, alopecia, pruritus, and hyperkeratosis. Septicemia and death may then eventuate (Pence and Ueckermann 2002). High mortality rates of up to 90% have been reported in a variety of European wild mammals (Oleaga et al. 2011; Devenish-Nelson et al. 2014).

In Australia, sarcoptic mange is reported in a range of native and feral mammals, as well as humans and domestic animals (Fraser et al. 2016). Wombats appear to be particularly susceptible, with a recent report describing an epizootic leading to a 94% decline in a bare-nosed wombat (Vombatus ursinus) population (Martin et al. 2018). The emergence of sarcoptic mange in geographically isolated populations of Australian marsupials is potentially explained by growing evidence that S. scabiei may have been introduced into Australia by European settlement in the 1800s (Skerratt 2005). The first evidence supporting this scenario arose from analysis of partial gene sequences from a small collection of marsupial S. scabiei samples, revealing close similarity to sequences from domestic animals and humans (Skerratt et al. 2002; Walton et al. 2004). Whole mitochondrial genome sequencing of several S. scabiei mites from koalas (Phascolarctos cinereus) and wombats confirmed these earlier observations, detecting genetically distinct S. scabiei mitochondrial DNA sequences in marsupials with some sharing closer genetic relationships to mites from hosts in other countries than to other marsupial S. scabiei sequences (Fraser et al. 2017). The lack of extensive population genetics data for S. scabiei in Australian fauna hinders further insight into spillover events and the potential sources of the infestation. In the current study, we begin the process of establishing a population genetic structure for S. scabiei in Australian animals, acquiring new mite sequences from a variety of hosts including dogs (Canis lupus familiaris), red foxes (Vulpes vulpes), koalas, and bare-nosed wombats across five states of Australia.

A total of 81 skin scrapings were collected from four Northern Territory (NT) dogs, four South Australian koalas, two Victorian foxes, and 11 Tasmanian and 45 New South Wales (NSW) bare-nosed wombats across several geographic locations. Collection of skin scrapings during routine veterinary diagnosis of mange-infested animals was approved by the Animal Research Committees at the University of the Sunshine Coast (approval AN5/16/43) and University of Tasmania (approval A0014670) and state permits from the Office of Environment & Heritage NSW National Parks & Wildlife Service (SL101719), Department of Primary Industries, Park, Water and Environment for Tasmania (approval FA15121), and Victorian Department of Environment and Primary Industries (10007943).

Scrapings were stored in 100% ethanol. The DNA extractions were performed on both pooled mites from a single skin scraping or on the complete skin scraping using a QIAamp DNA mini kit (Qiagen, Hilden, Germany). Conventional PCR and sequencing, targeting a 400 base pair fragment of the cox1 gene (Andriantsoanirina et al. 2015), were then performed. This gene was selected for molecular typing since previous analyses showed it was phylogenetically informative and mostly congruent with whole S. scabiei mitochondrial genome phylogenies (Fraser et al. 2016, 2017). Each 25 μL conventional PCR assay consisted of 1× Amplitaq Gold 360 Master Mix (Life Technologies Australia, Mulgrave, Victoria, Australia), 0.3 μM of forward and reverse primers, and 5 μL DNA template. Cycling conditions were 95 C for 10 min, 35 cycles of 95 C for 15 s, 51 C for 30 s, and 72 C for 1 min, followed by a final extension of 72 C for 7 min. Positivity was confirmed following agarose gel electrophoresis and ultraviolet transillumination. Amplicon sequencing was performed by Macrogen Korea (Seoul, Republic of Korea).

All sequences were aligned with ClustalW. Phylogenetic tree construction was performed by Mr. Bayes (Huelsenbeck and Ronquist 2001) using the HKY85 model, rooted with Otodectes cynotis (GenBank sequence no. KP676688) with 1,000 bootstrap replicates, implemented by Geneious 9.1.8 (Kearse et al. 2012). Run parameters included four Markov Chain Monte Carlo chains with a million generations with a sampling frequency of 200 generations and the first 100,000 trees discarded as burn-in. Phylogenetic metadata analysis was performed with Phandango (Hadfield et al. 2018).

The PCR amplification and cox1 gene sequencing resulted in 81 new Australian-derived S. scabiei sequences to add to analyses of 24 previously described sequences (Table 1). Sixteen unique cox1 haplotypes could be identified in the Australian hosts following cox1 sequence alignment, sharing between 93.3% and 99.7% sequencing similarity. These haplotypes could be broadly divided into five groups (Table 1) including 1) three novel S. scabiei cox1 haplotypes (H10, H11, and H16) in wombats from NSW; 2) two Australian S. scabiei cox1 haplotypes (H4, H5), representing S. scabiei sequences from NT dogs and Tasmanian wombats and identical to S. scabiei sequences previously only observed in other Australian animals; 3) four S. scabiei haplotypes (H2, H9, H12, and H14), representing sequences from dogs and wombats, that were identical to previously described sequences from Australian marsupials (i.e., wallabies and wombats) and dogs as well as hosts from other parts of the world; 4) haplotypes detected in humans in Australia and other parts of the world (H6, H7, H8, H15, and H16); and 5) two Australian-specific dog haplotypes (H1 and H3) previously identified by Walton et al. (2004).

Table 1

Detailed list of Sarcoptes scabiei cox1 sequence haplotypes from Australian animals identified in this study and from Australian marsupials as reported elsewhere; list of haplotype relationships to cox1 sequences detected in other parts of the world is also included.

Detailed list of Sarcoptes scabiei cox1 sequence haplotypes from Australian animals identified in this study and from Australian marsupials as reported elsewhere; list of haplotype relationships to cox1 sequences detected in other parts of the world is also included.
Detailed list of Sarcoptes scabiei cox1 sequence haplotypes from Australian animals identified in this study and from Australian marsupials as reported elsewhere; list of haplotype relationships to cox1 sequences detected in other parts of the world is also included.

To understand the distribution and relationships of these sequences further, we constructed a phylogenetic tree of all Australian available cox1 sequences (Fig. 1). In the case of bare-nosed wombats, the most comprehensively sampled host in this study, at least nine genetically distinct S. scabiei strains have now been detected, revealing an unexpectedly high level of genetic diversity of circulating S. scabiei. As seen in Table 1, the NSW wombat population appears to harbor at least six S. scabiei haplotypes (H9–H14) with three sequences unique to this population (H10, H11, and H13), while the other three haplotypes have been detected in other hosts globally (H12, H14) and/or in other marsupials in other states (H9). The observation that many of these wombat haplotypes could also be detected in koalas (indeed, we did not detect any koala-specific lineages) suggested that individual lineages of S. scabiei mites can readily infest a range of Australian marsupials.

Figure 1

Phylogenetic tree of Australian Sarcoptes scabiei cox1 sequences. Bayesian phylogenetic analysis of 105 cox1 sequences for Australian hosts only, rooted with Otodectes cynotis. Bootstrap values >80 are indicated. Haplotype, host, and location are indicated, corresponding to each node label using Phandango (Hadfield et al. 2018).

Figure 1

Phylogenetic tree of Australian Sarcoptes scabiei cox1 sequences. Bayesian phylogenetic analysis of 105 cox1 sequences for Australian hosts only, rooted with Otodectes cynotis. Bootstrap values >80 are indicated. Haplotype, host, and location are indicated, corresponding to each node label using Phandango (Hadfield et al. 2018).

In terms of evidence for disease spillover in Australia, we also detected at least one S. scabiei cox1 haplotype (H4) that was previously detected in dogs from the NT and, in our study, was shared by Victorian wombats, koalas, and foxes. This same haplotype could also be detected in South Australian koalas and, again, from dogs more recently sampled in the NT. Since this appears to be an Australian-specific lineage, we cannot yet deduce the directionality of this cross-host transmission; however, dogs have long been suspected as a potential source of marsupial S. scabiei infestation (Skerratt et al. 2002), and these new data, including the identification of this same sequence in foxes, support this hypothesis. Perhaps an even better case for canine reservoirs of marsupial infestation also lies in the detection of cox1 haplotype 2 (Table 1) in a dog in this study and marsupials from previous studies (Walton et al. 2004; Fraser et al. 2017), as well as a range of other dogs in the rest of the world.

The failure to separate the majority of Australian S. scabiei sequences from other global sequences, as well as the presence of at least one human-specific lineage (containing haplotypes H15 and H16), is further illustrated in Figure 2. Together these data support a hypothesis that Australian marsupials may be a sink for genetically diverse animal S. scabiei lineages introduced from the rest of the world, additionally evidenced by haplotypes H9, H12, and H14, which include domestic and nondomestic animals from outside Australia (Table 1). This hypothesis of multiple mange introduction events has also been implicated in North American bears (Peltier et al. 2017) and canids in Japan (Matsuyama et al. 2015).

Figure 2

Global Bayesian phylogenetic analysis for the cox1 gene of Sarcoptes scabiei. Phylogenetic assessment of Australian haplotypes and globally available cox1 sequences for S. scabiei, rooted with Otodectes cynotis. Bootstrap values >80 are shown. Haplotypes for Australian humans only, Australian dogs only, Australian animals only, shared global and Australian animal hosts, and shared global and Australian human hosts are indicated by the key.

Figure 2

Global Bayesian phylogenetic analysis for the cox1 gene of Sarcoptes scabiei. Phylogenetic assessment of Australian haplotypes and globally available cox1 sequences for S. scabiei, rooted with Otodectes cynotis. Bootstrap values >80 are shown. Haplotypes for Australian humans only, Australian dogs only, Australian animals only, shared global and Australian animal hosts, and shared global and Australian human hosts are indicated by the key.

This is the largest phylogenetic analysis of gene sequences derived from S. scabiei mites from Australian wildlife and provides significantly more data on the population genetic structure of this important human and animal pathogen in Australian animals. Based on the evidence in this study, previous studies of mange in Australian animals (Fraser et al. 2017), and studies from other wildlife hosts (Peltier et al. 2017), it is likely that some of this genetic diversity is likely due to multiple introductions of S. scabiei into native marsupials from canids (dogs and foxes) and possibly humans. Sympatric sampling studies of marsupials and potential animal reservoirs such as dogs and foxes will be required to confirm these suspected spillover events.

The authors would like to acknowledge Keeley Thomas and Holly Lubcke for their assistance with processing skin scrapings and Martina Jelocnik for her help with GenBank submission. This work was funded by the Holsworth Wildlife Research Endowment to T.A.F.

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