All relevant data are within the paper and its Supporting Information files. Introduction {#sec001} ============ Infectious disease surveillance and monitoring in wildlife is required by the Convention on International Trade in Endangered Species (CITES), as well as wildlife management authorities to document disease status in wildlife populations and to guide mitigation efforts that may control the wildlife trade or prevent wildlife from coming into contact with domestic livestock or people. A primary application of wildlife disease monitoring is to provide data on host-pathogen associations that can guide research, diagnostic test development, and surveillance in domestic and wildlife hosts. This is especially important for emerging zoonotic pathogens that cannot be easily diagnosed in wildlife, but where the role of domestic animals in human infections is unclear \[[@pone.0144063.ref001],[@pone.0144063.ref002]\]. Data on host-pathogen associations is also vital for the development of molecular epidemiology and disease control programs in wildlife populations as well as to identify priority species that may be threatened by emerging zoonotic pathogens in wildlife \[[@pone.0144063.ref003]--[@pone.0144063.ref006]\]. The study of diseases in wildlife is both challenging and rewarding. Wild animals can harbor novel pathogens or carry known pathogens, which can be difficult to identify when using non-molecular techniques, leading to under-diagnosis of emerging pathogens and misidentification of endemic pathogens \[[@pone.0144063.ref007]\]. In many cases the study of wildlife diseases can benefit from the use of well-developed disease and clinical biochemistry diagnostic testing techniques that are used routinely in domestic species \[[@pone.0144063.ref008]\]. However, many wildlife species are also highly-specialized (i.e. some endangered species) and often live in protected natural reserves (i.e. inaccessible locations), which hinders research access and increases the number of pathogens that are unseen \[[@pone.0144063.ref009],[@pone.0144063.ref010]\]. For pathogens to be identified and studied in wildlife, the development of robust sampling and testing methods is required \[[@pone.0144063.ref011]\]. Currently, sample collection is the most critical step in wildlife disease surveillance, as many diseases are known to cause only minimal clinical signs in the host \[[@pone.0144063.ref012]--[@pone.0144063.ref014]\]. For many pathogens, infection in wildlife often remains asymptomatic and/or non-lethal. For these diseases, sample collection from individuals that are free-ranging or live in remote locations may be required in order to fully characterize the host-pathogen association \[[@pone.0144063.ref015]\]. The use of passive sampling in wildlife disease surveillance provides an opportunity for the detection of pathogens that could go undetected if active surveillance is required \[[@pone.0144063.ref016]\]. Several different types of sampling strategies can be used in wildlife surveillance, depending on the pathogen and disease epidemiology. In many cases the use of a syndromic approach is best suited for the identification of pathogens in wildlife \[[@pone.0144063.ref014]\]. The use of this method minimizes the cost of identifying possible pathogens and avoids missing data due to the absence of symptoms in the host \[[@pone.0144063.ref017]\]. The use of syndromic approaches in wildlife disease surveillance has been performed in many wildlife studies, including those investigating emerging or re-emerging pathogens \[[@pone.0144063.ref018]--[@pone.0144063.ref023]\]. However, this approach has only been applied to diseases with clear clinical signs in domestic animals and is limited by the absence of diagnostic tests in wildlife. Disease surveillance for emerging zoonotic pathogens in wildlife needs to be broad, efficient, sensitive, and have little cost per sample to ensure efficient pathogen identification and surveillance. This process of surveillance will benefit from having a syndromic sampling strategy, which will maximize sampling efficiency and reduce the overall cost of the surveillance program by targeting only those animals that are infected. Currently, syndromic approaches for the surveillance of pathogens in wildlife are based on the combined use of disease surveillance (e.g. visual or clinical assessment) and non-invasive sampling techniques (e.g. feather and faeces sampling). The non-invasive approach for surveillance of wildlife diseases has several advantages including the ability to be applied to larger animal populations for extended periods of time, low risk to the sampled animal, minimal cost, and low operational bias (e.g. the ability to collect large amounts of data from a limited number of animals). In contrast to domestic animals that are regularly monitored during pregnancy and at breeding or milking facilities, rare or endangered wildlife species have fewer obvious time points at which to monitor their health status. Ideally, large wildlife populations are subject to long term observation through the use of passive non-invasive sampling techniques. To expand the utility of wildlife health monitoring, our research uses the domestic sheep (red deer), puma, and American black bear as models to: (1) investigate the feasibility and repeatability of a syndromic approach for the monitoring of endemic and novel disease agents in wildlife, and (2) investigate the effect of different sampling procedures on pathogen detection rate in wildlife. Pathogens were used as models due to their global importance and range of clinical symptoms in animals. The goals of this study were (1) to evaluate the use of faecal samples from live-captured free-ranging wildlife as a potential diagnostic tool for endemic and novel wildlife diseases, (2) to evaluate a non-invasive syndromic sampling technique for surveillance of pathogens in wildlife, and (3) to evaluate if repeated testing is possible in non-invasive fecal samples from free-ranging wildlife to monitor the health status of the population. The specific objectives of this study were to evaluate if: (1) multiple samples collected from a single individual could be pooled to increase diagnostic sensitivity, (2) faecal samples from live-captured wildlife could be used to detect pathogens that cause endemic diseases and/or novel agents that have been implicated in disease outbreaks, and (3) a non-invasive faecal sampling technique for wildlife diseases could be used for pathogen surveillance. A non-invasive method for pathogen surveillance in free-ranging wildlife would improve the ability to efficiently monitor wildlife diseases in remote locations and provide timely information about wildlife health status. Materials and Methods {#sec002} ===================== Ethics Statement {#sec003} ---------------- Scientific Research License Permits were obtained from New South Wales National Parks and Wildlife Service (NWS) for the capture, handling and sampling of wildlife in NSW. Procedures used in this study were approved by the Hunter New England Animal Ethics Committee (Permit Number: AEC10/14-15), the University of Sydney Animal Ethics Committee (Permit Number: L04/6-2009/3/4347), and the NSW NWS (Permit Number: P11/04). Study Area and Sampling Method {#sec004} ------------------------------ A total of 11 sites in NSW, Australia ([Table 1](#pone.0144063.t001){ref-type="table"}) were sampled between 2010 and 2013 ([Fig 1](#pone.0144063.g001){ref-type="fig"}) during wildlife disease surveillance studies of pathogens in free-ranging endangered and at-risk species \[[@pone.0144063.ref018]--[@pone.0144063.ref023]\]. The sites sampled varied in size, from 100 m^2^ to 1,000,000 m^2^ ([Table 1](#pone.0144063.t001){ref-type="table"}). Sites 1 and 2 were located at Healesville Sanctuary in Victoria; sites 3--5 were located at Cootamundra National Park in NSW; and sites 6--8 were located at Wollemi National Park, NSW. Sites 9--11 were located in the Greater Blue Mountains Area of NSW ([Fig 1](#pone.0144063.g001){ref-type="fig"}) near Katoomba, Australia. {#pone.0144063.g001} 10.1371/journal.pone.0144063.t001 ###### Sample sites, area, and management type. The data regarding the areas are from Hunter New England (HNE) and the management type is from NSW National Parks and Wildlife Services (NWS). {#pone.0144063.t001g} Site Area (ha) Management type Site Area (ha) Management type ------ ----------- ------------------------------------------------------------------------------------ ------ ----------- ------------------------------------------------------------------- S1 250 Grazing S11 350 Grazing S2 170 Beef cattle grazing