of Emerging Infectious Diseases in Wildlife Trade in Lao

10th APEIR Regional Meeting: The New Wave of Regional EID Research Partnership" Bali, Indonesia, 13-14 October 2016 Wildlife trade project in Lao PDR Progress of the project implementation on Surveillance of Emerging Infectious Diseases in Wildlife Trade in Lao PDR

Zoonotic diseases Zoonotic diseases in people are increasing; responsible for ~ 70% of emerging human pathogens. 70%of Zoonotic diseases origins from wildlife In Lao PDR Scrub typhus and Leptospirosis are known to be high. 15% of adults admitted with fever at Mahosot Hospital have evidence of scrub typhus.

Zoonotic diseases

Zoonotic disease in Lao PDR In Lao PDR: Development; agriculture, road, mining, dam, logging Wildlife trade is common and high volume - Traditional belief that eating wildlife is healthier - Markets are crowded with people and there are low hygiene practices - High risk species High risk for zoonotic diseases

3 Main Objectives: Zoonostic Diseases Active surveillance in wildlife trade in Lao PDR To understand the disease circulation and introduction in wildlife To study risk factor and KAP of relevant of stakeholder involve with wildlife trade in order to develop the measure to control and prevention on wildlife disease prevention and control Product the public awareness material and disseminate in order for behavior changing to appropriate action in contacting or consuming of wildlife as well as To raise (improve) awareness of specific stakeholders ( vender, favorite wildlife consuming, and hunter )of how to prevent diseases which originate from wildlife and also contribute to wildlife conservation

Project arrangement Memorandum of Grand condition was signed by APEIR and DLF dated on 09 August 2014 Project to be authorized for no cost extension on Project was approved for ethical clearance Set up research team work and advisory committee

PROCESS Wildlife sampling Lab analysis Information sharing Intervention and Prevention

PROCEDURE DETAILS Wildlife sampling Lab testing Samples: Urogenital swab, blood, urine, kidney, spleen, liver Animals/Vectors: livestock, rodents, birds, bats, ticks, chiggers Transport mediums: RNA-later, Viral Transport medium, alcohol, plain cryovials LACANET-specific extraction protocol that extracts both DNA & RNA simultaneously T4/MS2 phage added as an internal control for PCR inhibition; dilution of samples Conventional and Real-time PCRs to detect different pathogens Sequencing for speciation

Activities for study Wildlife Trade In Lao PDR 1 wildlife trade survey on species, volume 2 Sampling and testing for Zoonotic pathogen 3 KAP study for Vendor and Consumer

Study site From late 2014- early 2016, Wildlife trade observed at 21 sites, in 14 districts 7 Provinces between 16 markets 5 roadside markets/stalls

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 Species 1 Pallas s squirrel 48 2 Grey-bellied squirrel 30 3 Red-cheeked squirrel 29 4 Indochinese ground squirrel 21 5 Common palm civet 19 6 Variable squirrel 19 7 Hoary bamboo rat 19 8 Small flying squirrel 10 9 Indian giant flying squirrel 7 10 Small Asian mongoose 3 11 Large bamboo rat 3 12 Bamboo rat 2 13 Lesser giant flying squirrel 2 14 Large-spotted civet 1 15 Red muntjac 1 16 Red-cheeked flying squirrel 1 17 Rat species TBD 1 18 Greater long-tongued nectar bat 1 19 Phayrei s flying squirrel 1 20 Black giant squirrel 1 21 Leopard cat 1 22 Yellow-throated marten 1 23 Small-toothed palm civet 1 24 Burmese hare 1 25 Inornate squirrel 1

TARGET PATHOGENS BACTERIA o Orientia tsutsugamushi (47kDa) o Rickettsia spp. (17kDa) o Leptospira spp. (rrs) o Anaplasma phagocytophilum (msp2) o Ehrlichia chaffeensis (ECH) o Coxiella burnetii (IS1111) o Neorickettsia spp. (16s rrna) VIRUSES o Flavivirus o Hantavirus o Dengue virus o Zika virus o Rabdo virus o Coronavius parasit o trichenella

Key results

Wildlife Volume IDRC; 11,664 carcasses (108 species from 3,846 individuals identified) or 5,750 kg biomass of wildlife observed for sale over 145 surveys. 846 alive or fresh dead mammals (approximately 693 kg) were observed for sale, including mammals from 9 taxonomic families that have been previously documented to be capable of hosting 36??? zoonotic pathogens. This represents only fraction of trade.

Some information from other study on risk factors in wildlife market Opportunities for pathogen transmission in Markets

Wildlife Trade In Lao PDR Zoonotic diseases Wildlife trade project : 1, 096 samples from 530 animals were collected from wildlife markets and roadside markets/stalls to test for priority zoonotic pathogens 127 positive samples o o o o o 73 coronaviruses in bats, rodents and wild viverids 44 Leptospira sp in rodents 6 Rickettsia sp in rodents 1 Lactococcus garvieae in 1 Kurthia spp o 1 Ehrlichia spp. TC251-2; 1 o 1 Anaplasma marginale in Muntjac On-going work to confirm and characterize these pathogens

Capacity building 3 participants from National Animal Health laboratory were attended the training course for the Laboratory Diagnostic and Biosafety Training at the Faculty of Veterinary Science of Mahodol University, Thailand, from 31 march to 3 April 2014 Opened the inception workshop to introduce the project activities and plan to the provincial discussion maker from Agriculture and Forestry department, Public health department, Natural resources and development from the province of Champasack, Xaysomboun, Borikhamxay, and Savannakhet on 12 May 2014 at Champasack province, Lao PDR

13-14 May 2014 the training on wildlife disease surveillance and monitoring techniques 12-14 July 2014 Lao wildlife trade project organized the training on Biosecurity and BSLIII Laboratory Management

3 Main Objective: To estimate the degree of zoonotic pathogens in wildlife trade within the region surveillance activities To identify the risk situation and risk behaviors of specific stakeholders that promote disease transmission from wildlife trade to humans including the existence of current zoonotic prevention and outbreak response measures making behaviour study in human To raise (improve) awareness of specific stakeholders of how to prevent diseases which originate from wildlife and also to raise awareness for wildlife conservation efforts produce the public awareness material

Form A: Market General Observation FormB: Products sold by individual vendor Form C: Market Actors Observation Form D. Small market/roadside vendor observation Form E: Market Snapshot to be used for roadside vendors and non-main markets. KAP survey form /develop by Dr wittawat and his team

Sharing results to the villager

Limitation Lack of sequencing capacity. Most of sample will send for sequencing need cost and document support Limit of expertise who are able to supervise in KAP analysis in Lao PDR Changing of new mechanism for project coordination and facilitation in regional level is one cause of project implementation to be delayed.

FUTURE PLAN o o o o o To coordinate with Pasteur institute in Cambodia for conducting the Sequencing of presumptive positive sample Leptospira spp. Rickettsia spp. PossibleNeorickettsia/Anaplasma/Ehrlichia spp. Corona virus continue to conduct the KAP survey with university of Lao in wildlife market Organize meeting with stakeholder to share the result from research Publication and Sharing the results to the international meeting and congress Enhance the Capacity building on sequencing technique to be exist in Lao PDR

Conclusion Long term data on wildlife trade provide important insight into zoonotic disease risk Building durable wildlife surveillance mechanisms is essential for Lao PDR s capacity to detect priority and emergent zoonosis Strengthening an often neglected part of One Health

Acknowledgement APEIR/IDRC Department of Livestock and Fisheries/ National Animal Health Laboratory Provincial agriculture and Forestry department National University of Lao Ministry of natural resource and environment