A One Health approach to investigating the health and prevalence of zoonotic pathogens in snow leopards, sympatric wildlife, domestic animals and humans in the South Gobi Desert in Mongolia

The endangered Snow leopard (Panthera uncia) inhabits the high mountain regions through central Asia and is subjected to numerous threats including poaching for traditional Chinese medicine, retribution killing for preying on domestic stock, and habitat fragmentation. However the occurrence and impact of disease on snow leopard populations is unknown. As emerging infectious diseases of wildlife can be an insidious yet important cause of population decline due to mortality or reproductive failure, my study aimed initially to gain knowledge of pathogens circulating among wild and domestic hosts in this region. I used a broad One Health approach to survey a range of species to collect data on disease occurrence that would be useful in improving human and livestock health, as well as snow leopard conservation. This study is set in the Tost Mountains of the South Gobi Desert of Mongolia and was prompted due to the unexplained deaths of four snow leopards detected within a short timeframe during an ecological study by members of the Snow Leopard Trust. However, investigating disease occurrence in remote, rare and endangered species is a challenge due to inaccessibility of sites, difficulty of capture, and processing samples without facilities. A One Health approach uses multidisciplinary expertise such as ecological, medical and veterinary, to understand host, pathogen and environmental disease factors. This approach is especially useful for diseases that transfer between people, domestic animals and wildlife. As snow leopards are a rare and elusive species, my surveys were aimed at assessing pathogens circulating in snow leopards as well as in sympatric wild and domestic animals. I collected samples from the following hosts: snow leopards – the target species; rodents which are ubiquitous over the study area and are a suitable sentinel species; ibex which are a native ungulate and natural prey species of the snow leopard; domestic goats which are also a prey species of the snow leopard; free-ranging domestic dogs which interact with the goats. The local indigenous people interact with all these species including snow leopards, mostly via retribution killing. Water samples were also collected from waterholes and wells, which are communal meeting places as drinking sources for all species, hence enabling pathogen exchange. Samples collected included blood samples, faecal samples or rectal swabs and ectoparasites if present. These samples were transported to laboratories in Sweden and Belgium where I conducted diagnostic assays for zoonotic pathogens that are present in other regions of Mongolia and impact the health of humans and animals. I used enzyme- linked immune assay (ELISA), polymerase chain reaction (PCR) and next-generation sequencing (NGS) for pathogens including Coxiella burnetii, Toxoplasma gondii, Leptospira spp., Brucella spp., Yersinia pestis and tick borne encephalitis virus. Serovars of Leptospira were elucidated using microscopic agglutination tests (MAT). The dog blood samples were also tested for canine distemper virus. Ticks, faeces, rectal swabs and water were tested for bacteria, Echinococcus, Giardia and Cryptosporidium using PCR and NGS. Health records for humans and animals in the region were not available so, in addition to testing animal samples, I used questionnaire surveys to obtain information on perceptions of the herders concerning health of their families, their domestic animals and wildlife. Questions also assessed preventative health management and treatments used. Over three field trips I caught and sampled twenty snow leopards, 177 rodents (8 species), 41 dogs and 270 goats. I also sampled 11 waterholes/wells, and preserved 18 ticks, hundreds of fleas and collected faecal samples from ibex. Most animals that were sampled and examined clinically appeared in good health, but the serosurvey revealed a moderate to high level of exposure to serious pathogens: C. burnetii, T. gondii and Leptospira spp. There were no published reports of human infections with these pathogens in the study area, which is likely due to a lack of testing. Snow leopards had the highest prevalence of C. burnetii antibodies (25%), followed by rodents (16%), dogs (10%) and goats (9.5%). Goats had the highest prevalence of T. gondii antibodies (90%), dogs (66%), snow leopards (20%) and rodents (16%). Rodents had the highest prevalence of Leptospira spp. (34%), followed by snow leopards (20%) and dogs (5%). Serovars interrogans Australis was identified in the rodents and snow leopards and interrogans Ictohaemorrhagiae was identified in the rodents and dogs. Other serovars were also present from the results of the ELISA but did not match those listed in the MAT panel, so could not be identified. Goats were not tested for infection with leptospirosis. Brucella was not identified in the goats even though it occurs at high prevalence in stock in the rest of Mongolia where it is a large health and economic concern. In rodents, the zoonotic Puumala and Seoul hantavirus were identified for the first time in Mongolia. Analysis of data from rodents showed the pathogens detected (C. burnetii, T.gondii, Hanta virus and Leptospira spp.) differed significantly in prevalence, with a strong year effect driven mainly by Leptospira, which increased in prevalence across the three year study period. Toxoplasma gondii differed slightly in prevalence among rodent species. There was no significant difference in prevalence of interaction of pathogens among years or rodent species. Poor health was detected in goats with 10 out of the 14 goats tested via haematology and biochemistry being anaemic with haematocrits less than 20%. Haematology and biochemistry values for the other animal species appeared normal. I established haematology and biochemistry reference tables for two rodent species - red-cheeked ground squirrels and jerboas. Water samples were negative for serious pathogens. Fleas were negative for Yersinia pestis. However, ticks were positive for several genera of potential zoonoses, including Anaplasma, Bacillus, Coxiella, Clostridia, Francisella, Rickettsia, Staphylococcus, Streptococcus and Yersinia. Faecal samples were also positive for genera of potentially zoonotic bacteria including those listed above plus Bacteroides, Bordetella, Campylobacter and Enterococcus. Results from the two questionnaire surveys revealed the main reported illness in people were colds and flu. However, the local doctor also reported hepatitis as common. She also said that the local people contracted brucellosis whereas I did not identify this pathogen in their livestock. The herders thought their main loss of stock was from predation, with wolves identified as the main predator and snow leopards as the second. Other causes of stock loss perceived as important were adverse climatic conditions such as drought or severe winters while infectious disease was not a concern. Results from these surveys also highlighted gaps in health care for humans and livestock, especially around vaccination and parasite treatments. In summary, I found that snow leopards and other wild and domestic animals within the study area tested positive for previous exposure to several important zoonotic pathogens. These pathogens were likely circulating among species via contamination of pasture and via predation and have potential to cause illness and reproductive loss. However, I detected no adverse effects on the health of the animals due to infection with these pathogens, and observed no related mortality or illness during my field trips. Hence the deaths of the four snow leopards that were the impetus for my study have not been explained, and monitoring and surveillance of this population should continue. My findings on wildlife and domestic animal pathogens have relative importance to improving productivity of livestock and the health of the nomadic herders. I recommend improving the health of goats through vaccination and anti-parasite programmes, which will improve their fecundity and survival and thus increase herder income. These programmes will also have flow-on effects to improve the health of the native ungulates that share the grazing areas by decreasing the risk of pathogen transfer between them and also to the snow leopards that prey on them. Demonstrating the importance of herd health may also help mitigate herder wildlife conflict as increased productivity could decrease the perceived importance of predation on herd numbers. Coxiella burnetii and Leptospires spp are a likely cause of illness in people, despite the lack of reported diagnoses. As rodents had a moderate prevalence of all pathogens tested and inhabit the gers of the local people, it is important to raise awareness of the risk of pathogen transfer to people via rodent excrement contaminating stored food and eating utensils. Risk of human exposure to pathogens during goat slaughter can also be reduced via improved hygiene practices. By identifying pathogens with broad host ranges in a variety of species in this remote mountainous region, my study provides the basis for understanding health risks to wildlife, domestic animals and humans. Consideration of likely transmission routes for pathogens between species can inform current recommendations to improve health, productivity and hence conservation, of the endangered snow leopard – The Ghost of the Mountain

Increasing risks for emerging infectious diseases within a rapidly changing High Asia

The cold and arid mountains and plateaus of High Asia, inhabited by a relatively sparse human population, a high density of livestock, and wildlife such as the iconic snow leopard Panthera uncia, are usually considered low risk for disease outbreaks. However, based on current knowledge about drivers of disease emergence, we show that High Asia is rapidly developing conditions that favor increased emergence of infectious diseases and zoonoses. This is because of the existing prevalence of potentially serious pathogens in the system; intensifying environmental degradation; rapid changes in local ecological, socio-ecological, and socio-economic factors; and global risk intensifiers such as climate change and globalization. To better understand and manage the risks posed by diseases to humans, livestock, and wildlife, there is an urgent need for establishing a disease surveillance system and improving human and animal health care. Public health must be integrated with conservation programs, more ecologically sustainable development efforts and long-term disease surveillance

Health and zoonotic infections of snow leopards Panthera unica in the South Gobi desert of Mongolia

Background: Snow leopards, Panthera uncia, are a threatened apex predator, scattered across the mountains of Central and South Asia. Disease threats to wild snow leopards have not been investigated.Methods and Results: Between 2008 and 2015, twenty snow leopards in the South Gobi desert of Mongolia were captured and immobilised for health screening and radio-collaring. Blood samples and external parasites were collected for pathogen analyses using enzyme-linked immunosorbent assay (ELISA), microscopic agglutination test (MAT), and next-generation sequencing (NGS) techniques. The animals showed no clinical signs of disease, however, serum antibodies to significant zoonotic pathogens were detected. These pathogens included, Coxiella burnetii, (25% prevalence), Leptospira spp., (20%), and Toxoplasma gondii (20%). Ticks collected from snow leopards contained potentially zoonotic bacteria from the genera Bacillus, Bacteroides, Campylobacter, Coxiella, Rickettsia, Staphylococcus and Streptococcus.Conclusions: The zoonotic pathogens identified in this study, in the short-term did not appear to cause illness in the snow leopards, but have caused illness in other wild felids. Therefore, surveillance for pathogens should be implemented to monitor for potential longer- term disease impacts on this snow leopard population

The prevalence of rodent-borne zoonotic pathogens in the South Gobi desert region of Mongolia.

peer reviewedThe alpine ecosystems and communities of central Asia are currently undergoing large-scale ecological and socio-ecological changes likely to affect wildlife-livestock-human disease interactions and zoonosis transmission risk. However, relatively little is known about the prevalence of pathogens in this region. Between 2012 and 2015 we screened 142 rodents in Mongolia's Gobi desert for exposure to important zoonotic and livestock pathogens. Rodent seroprevalence to Leptospira spp. was >1/3 of tested animals, Toxoplasma gondii and Coxiella burnetii approximately 1/8 animals, and the hantaviruses being between 1/20 (Puumala-like hantavirus) and <1/100 (Seoul-like hantavirus). Gerbils trapped inside local dwellings were one of the species seropositive to Puumala-like hantavirus, suggesting a potential zoonotic transmission pathway. Seventeen genera of zoonotic bacteria were also detected in the faeces and ticks collected from these rodents, with one tick testing positive to Yersinia. Our study helps provide baseline patterns of disease prevalence needed to infer potential transmission between source and target populations in this region, and to help shift the focus of epidemiological research towards understanding disease transmission among species and proactive disease mitigation strategies within a broader One Health framework

Effects of antiplatelet therapy on stroke risk by brain imaging features of intracerebral haemorrhage and cerebral small vessel diseases: subgroup analyses of the RESTART randomised, open-label trial

Background Findings from the RESTART trial suggest that starting antiplatelet therapy might reduce the risk of recurrent symptomatic intracerebral haemorrhage compared with avoiding antiplatelet therapy. Brain imaging features of intracerebral haemorrhage and cerebral small vessel diseases (such as cerebral microbleeds) are associated with greater risks of recurrent intracerebral haemorrhage. We did subgroup analyses of the RESTART trial to explore whether these brain imaging features modify the effects of antiplatelet therapy

Effects of antiplatelet therapy after stroke due to intracerebral haemorrhage (RESTART): a randomised, open-label trial

Background: Antiplatelet therapy reduces the risk of major vascular events for people with occlusive vascular disease, although it might increase the risk of intracranial haemorrhage. Patients surviving the commonest subtype of intracranial haemorrhage, intracerebral haemorrhage, are at risk of both haemorrhagic and occlusive vascular events, but whether antiplatelet therapy can be used safely is unclear. We aimed to estimate the relative and absolute effects of antiplatelet therapy on recurrent intracerebral haemorrhage and whether this risk might exceed any reduction of occlusive vascular events. Methods: The REstart or STop Antithrombotics Randomised Trial (RESTART) was a prospective, randomised, open-label, blinded endpoint, parallel-group trial at 122 hospitals in the UK. We recruited adults (≥18 years) who were taking antithrombotic (antiplatelet or anticoagulant) therapy for the prevention of occlusive vascular disease when they developed intracerebral haemorrhage, discontinued antithrombotic therapy, and survived for 24 h. Computerised randomisation incorporating minimisation allocated participants (1:1) to start or avoid antiplatelet therapy. We followed participants for the primary outcome (recurrent symptomatic intracerebral haemorrhage) for up to 5 years. We analysed data from all randomised participants using Cox proportional hazards regression, adjusted for minimisation covariates. This trial is registered with ISRCTN (number ISRCTN71907627). Findings: Between May 22, 2013, and May 31, 2018, 537 participants were recruited a median of 76 days (IQR 29–146) after intracerebral haemorrhage onset: 268 were assigned to start and 269 (one withdrew) to avoid antiplatelet therapy. Participants were followed for a median of 2·0 years (IQR [1·0– 3·0]; completeness 99·3%). 12 (4%) of 268 participants allocated to antiplatelet therapy had recurrence of intracerebral haemorrhage compared with 23 (9%) of 268 participants allocated to avoid antiplatelet therapy (adjusted hazard ratio 0·51 [95% CI 0·25–1·03]; p=0·060). 18 (7%) participants allocated to antiplatelet therapy experienced major haemorrhagic events compared with 25 (9%) participants allocated to avoid antiplatelet therapy (0·71 [0·39–1·30]; p=0·27), and 39 [15%] participants allocated to antiplatelet therapy had major occlusive vascular events compared with 38 [14%] allocated to avoid antiplatelet therapy (1·02 [0·65–1·60]; p=0·92). Interpretation: These results exclude all but a very modest increase in the risk of recurrent intracerebral haemorrhage with antiplatelet therapy for patients on antithrombotic therapy for the prevention of occlusive vascular disease when they developed intracerebral haemorrhage. The risk of recurrent intracerebral haemorrhage is probably too small to exceed the established benefits of antiplatelet therapy for secondary prevention

Effects of antiplatelet therapy after stroke due to intracerebral haemorrhage (RESTART): a randomised, open-label trial

Background: Antiplatelet therapy reduces the risk of major vascular events for people with occlusive vascular disease, although it might increase the risk of intracranial haemorrhage. Patients surviving the commonest subtype of intracranial haemorrhage, intracerebral haemorrhage, are at risk of both haemorrhagic and occlusive vascular events, but whether antiplatelet therapy can be used safely is unclear. We aimed to estimate the relative and absolute effects of antiplatelet therapy on recurrent intracerebral haemorrhage and whether this risk might exceed any reduction of occlusive vascular events. Methods: The REstart or STop Antithrombotics Randomised Trial (RESTART) was a prospective, randomised, open-label, blinded endpoint, parallel-group trial at 122 hospitals in the UK. We recruited adults (≥18 years) who were taking antithrombotic (antiplatelet or anticoagulant) therapy for the prevention of occlusive vascular disease when they developed intracerebral haemorrhage, discontinued antithrombotic therapy, and survived for 24 h. Computerised randomisation incorporating minimisation allocated participants (1:1) to start or avoid antiplatelet therapy. We followed participants for the primary outcome (recurrent symptomatic intracerebral haemorrhage) for up to 5 years. We analysed data from all randomised participants using Cox proportional hazards regression, adjusted for minimisation covariates. This trial is registered with ISRCTN (number ISRCTN71907627). Findings: Between May 22, 2013, and May 31, 2018, 537 participants were recruited a median of 76 days (IQR 29–146) after intracerebral haemorrhage onset: 268 were assigned to start and 269 (one withdrew) to avoid antiplatelet therapy. Participants were followed for a median of 2·0 years (IQR [1·0– 3·0]; completeness 99·3%). 12 (4%) of 268 participants allocated to antiplatelet therapy had recurrence of intracerebral haemorrhage compared with 23 (9%) of 268 participants allocated to avoid antiplatelet therapy (adjusted hazard ratio 0·51 [95% CI 0·25–1·03]; p=0·060). 18 (7%) participants allocated to antiplatelet therapy experienced major haemorrhagic events compared with 25 (9%) participants allocated to avoid antiplatelet therapy (0·71 [0·39–1·30]; p=0·27), and 39 [15%] participants allocated to antiplatelet therapy had major occlusive vascular events compared with 38 [14%] allocated to avoid antiplatelet therapy (1·02 [0·65–1·60]; p=0·92). Interpretation: These results exclude all but a very modest increase in the risk of recurrent intracerebral haemorrhage with antiplatelet therapy for patients on antithrombotic therapy for the prevention of occlusive vascular disease when they developed intracerebral haemorrhage. The risk of recurrent intracerebral haemorrhage is probably too small to exceed the established benefits of antiplatelet therapy for secondary prevention

Effects of antiplatelet therapy after stroke due to intracerebral haemorrhage (RESTART): a randomised, open-label trial

BACKGROUND: Antiplatelet therapy reduces the risk of major vascular events for people with occlusive vascular disease, although it might increase the risk of intracranial haemorrhage. Patients surviving the commonest subtype of intracranial haemorrhage, intracerebral haemorrhage, are at risk of both haemorrhagic and occlusive vascular events, but whether antiplatelet therapy can be used safely is unclear. We aimed to estimate the relative and absolute effects of antiplatelet therapy on recurrent intracerebral haemorrhage and whether this risk might exceed any reduction of occlusive vascular events. METHODS: The REstart or STop Antithrombotics Randomised Trial (RESTART) was a prospective, randomised, open-label, blinded endpoint, parallel-group trial at 122 hospitals in the UK. We recruited adults (≥18 years) who were taking antithrombotic (antiplatelet or anticoagulant) therapy for the prevention of occlusive vascular disease when they developed intracerebral haemorrhage, discontinued antithrombotic therapy, and survived for 24 h. Computerised randomisation incorporating minimisation allocated participants (1:1) to start or avoid antiplatelet therapy. We followed participants for the primary outcome (recurrent symptomatic intracerebral haemorrhage) for up to 5 years. We analysed data from all randomised participants using Cox proportional hazards regression, adjusted for minimisation covariates. This trial is registered with ISRCTN (number ISRCTN71907627). FINDINGS: Between May 22, 2013, and May 31, 2018, 537 participants were recruited a median of 76 days (IQR 29-146) after intracerebral haemorrhage onset: 268 were assigned to start and 269 (one withdrew) to avoid antiplatelet therapy. Participants were followed for a median of 2·0 years (IQR [1·0- 3·0]; completeness 99·3%). 12 (4%) of 268 participants allocated to antiplatelet therapy had recurrence of intracerebral haemorrhage compared with 23 (9%) of 268 participants allocated to avoid antiplatelet therapy (adjusted hazard ratio 0·51 [95% CI 0·25-1·03]; p=0·060). 18 (7%) participants allocated to antiplatelet therapy experienced major haemorrhagic events compared with 25 (9%) participants allocated to avoid antiplatelet therapy (0·71 [0·39-1·30]; p=0·27), and 39 [15%] participants allocated to antiplatelet therapy had major occlusive vascular events compared with 38 [14%] allocated to avoid antiplatelet therapy (1·02 [0·65-1·60]; p=0·92). INTERPRETATION: These results exclude all but a very modest increase in the risk of recurrent intracerebral haemorrhage with antiplatelet therapy for patients on antithrombotic therapy for the prevention of occlusive vascular disease when they developed intracerebral haemorrhage. The risk of recurrent intracerebral haemorrhage is probably too small to exceed the established benefits of antiplatelet therapy for secondary prevention. FUNDING: British Heart Foundation

Health and zoonotic Infections of snow leopards Panthera unica in the South Gobi desert of Mongolia

Background: Snow leopards, Panthera uncia, are a threatened apex predator, scattered across the mountains of Central and South Asia. Disease threats to wild snow leopards have not been investigated. Methods and Results: Between 2008 and 2015, twenty snow leopards in the South Gobi desert of Mongolia were captured and immobilised for health screening and radio-collaring. Blood samples and external parasites were collected for pathogen analyses using enzymelinked immunosorbent assay (ELISA), microscopic agglutination test (MAT), and nextgeneration sequencing (NGS) techniques. The animals showed no clinical signs of disease, however, serum antibodies to significant zoonotic pathogens were detected. These pathogens included, Coxiella burnetii, (25% prevalence), Leptospira spp., (20%), and Toxoplasma gondii (20%). Ticks collected from snow leopards contained potentially zoonotic bacteria from the genera Bacillus, Bacteroides, Campylobacter, Coxiella, Rickettsia, Staphylococcus and Streptococcus. Conclusions: The zoonotic pathogens identified in this study, in the short-term did not appear to cause illness in the snow leopards, but have caused illness in other wild felids. Therefore, surveillance for pathogens should be implemented to monitor for potential longerterm disease impacts on this snow leopard population