2. Antimicrobial Resistance1. AMR: the global issues for health2. AMR in E. coli of the endangered bird in

Okinawa Island, Japan

AMR: the global issues for health

Berkner et al. (2014)

Pathogen flow among wild and domestic host-pathogen systems: spill-over & spill-back (Thompson et al., 2009)http://www.who.int/foodsafety/areas_work/antimicrobial-resistance/tripartite/en/

Pathway of antibiotics for human and veterinary use in the environment

E. coli as an indicator of AMR Why Escherichia coli?

• Wide range of distribution: human, livestock, wildlife and environment

• Easy to detect comparing with the other bacteria• Many back ground medical data and information

http://www.ecl-lab.com/en/ecoli/index.asp

AMR in E. coli of the endangered bird in Okinawa Island, Japan

Okinawa rail (Gallirallus okinawae)

The evergreen laurel forest zone called “Yanbaru” located in northern part of Okinawa

Island where many endangered endemic species inhabit

Development threats to natural environments

Some endangered species use stock farms as a new habitat

：The installation site of cameras and their direction

Camera trapping Trail camera (Ltl Acorn LTL 5310a) Relative Abundance Index （RAI）

=（effective shooting numbers ÷ (number of cameras × operating days) × 30

Installation condition 1-2m off the ground Angle of depression: 0-30°

60m

Surveillance of wildlife in a stock farm

Surveillance of AMR in wildlife Areas of surveillance

• Kunigami village, Okinawa Main Island, located at 26°45’ N and 128°17’ E

• Forest areas (FA) and Human habitation (HH) Samples

• Fecal samples from the Okinawa rail: 48 samples• Environmental samples (soil and water) from

the forest area of “Yanbaru”: 80 samples

: Forest area (FA): Human habitation (HH): Stock farm: Pref. road 70: Pref. road 58

Sites of collecting fecal samples: Forest area: Human habitation: Stock farm: Pref. road 70: Pref. road 58: Sampling site

FA

HH

Google maps of sampling points at the northern part of Okinawa Island Feces of the Okinawa rail on the Pref. road

Detection of AMR E. coli from feces

Human habit.

: Forest area: Human habitation: Stock farm: Pref. road 70: Pref. road 58: E. coli: AMR E. coli

57.7%15 SPL

42.3%11 SPL

72.7%16 SPL

27.3%6 SPL

Prevalence of E. coli

Prevalence of E. coli

20.0%3 SPL

80.0%12 SPL

32.1%5 SPL 68.8%

11 SPL

Prevalence of AMR E. coli

Forest area

Prevalence of AMR E. coli

: HH: Stock farm: Pref. road 70: Pref. road 58: Soil sampling site: Water sampling site

Site Soil（No of SPL）

Water（No. of SPL）

Total(No. of SPL)

FA 33 14 47

HH 21 13 34

HH

Sites of collecting environmental samples

Human habit.

Forest area : Human habitation: Stock farm: Pref. road 70: Pref. road 58: E. coli: AMR E. coli

16.6%2 SPL

66.7%10 SPL

8.3%1 SPL

91.7%11 SPL

25.5%12 SPL

74.5%35 SPL

36.4%12 SPL63.4%

21 SPL

Detection of AMR bacteria from environmental samples

Prevalence of E. coli

Prevalence of E. coli

Prevalence of AMR E. coli

Prevalence of AMR E. coli

: Fecal sample collected at the forest area

: Environmental sample collected at the forest area

Genotype of E. coli from samples

: Fecal sample collected at the stock farmSame genotype btw two sites 5km apart

MIC（µg/ml） OTC NA OFLX CP ST CET

Max.Min.

1281

>256-

>32-

>256-

>32-

32-

Abbreviations of antimicrobial agentsABPC PIPC KM OTC NA OFLX CP ST CET

FA 0 0 0 3 1 1 2 1 1HH 4 3 1 9 2 2 2 4 5

MIC（µg/ml） ABPC PIPC KM OTC OFLX CP NA ST CET

Max.Min.

>256-

>25632

>256-

>256128

>32-

>256-

>256-

>32-

>2568

Number of AMR E. coli detected from FA and HH

Minimum inhibitory concentration (MIC) score

Red: synthetic antibacterial agents

Resistance to antimicrobial agents

3. Avian Influenza1. Avian Influenza in Asian countries2. Avian Influenza in wild birds of Japan3. Molecular surveillance of avian influenza virus,

using waterfowl fecal samples

High & Low Pathogenic Avian Influenza outbreak The status of the outbreaks in poultries of HPAI & LPAI incidents of the world

HPAILPAI

As of 22 May 2017

High & Low Pathogenic AI in Asian Countries The status of occurrence of HPAI/LPAI in Asian regions from October 2016 to May 2017

As of 17 May 2017●chickens ▲wild birdsRed: HPAI Blue: LPAI

In Indonesia ⑱, avian influenza outbreak has continuously occurred.

Avian Influenza in wild birds of JapanBackground of coping with HPAI

surveillance in wild birds in Japan• 2004 infected crows were confirmed around

the chicken farms which had HPAI outbreaks (secondary infection) the first time in 79 years

• 2007 One Mountain hawk-eagle• 2008 Whooper swans• 2008 Manual of technique and coping with

HPAI in wild birds for local governmental agencies was published by Ministry of Environment→ Start nation-wide surveillance

• 2010-2011 Outbreaks in wild birds (15 species, 60 birds), domestic birds, and some endangered species as Hooded cranes and White-naped cranes in Izumi City, Kagoshima Prefecture

• 2011-2012 and 2012-2013 No HPAI in wild birds

• 2014-2015 H5N8 outbreaks • 2016-2017 H5N6 detected from 218 samples

consist of wild birds (22 species), 82 zoo birds (5 species), 5 fecal samples from wild duck and 3 water samples of roosting sites

HPAI (H5N6) outbreak in wild birds and poultry farm in Japan from 2016 to 2017• 218 cases of wild birds in 22 prefectural and

city governments - the highest number in 9years

• 12 of cases of poultry farms in 9 prefectural and city governments

: wild birds: poultry farm

Molecular surveillance of avian influenza virus using waterfowl fecal samples

by courtesy of Dr. Onuma of National Institute for Environmental Studies

AIV gene Positive335 samples (1.9%)

RT-LAMP method (EIKEN)

AIV gene detectionSpecies identification

221 samples

DNA barcoding and information of direct observation

Fecal sample collection in 53 sites covering all over Japan (2008-2015)

Total: 17,540 samples

Material and Methods

Temporal changes of RT-LAMP positive rate Seasonality of HPAI virus positive in wild birds from 2008 to 2015

• High positive rate in October to December• Positive rate declined in April to May

Species identification using fecal samples

Risk Index0.9‐1.00.8‐0.90.7‐0.80.6‐0.70.5‐0.60.4‐0.50.3‐0.40.2‐0.30.1‐0.20.0‐0.1Not analyzed

(Moriguchi et. al., 2013)

Risk analysis for avian influenza in wild birdsIndependent variables Contribution

Dabbling duck population  49.4 ± 5.5 

Urban area  12.5 ± 3.5 

Altitude 8.2 ± 2.3 

Diving duck population  3.5 ± 3.5 

Lake area  2.2 ± 1.5 

Farmland area  0.9 ± 1.2 

Poultry density  0.5 ± 0.5

Mean precipitation in winter  0.3 ± 1.0 

Min temperature in winter  0.1 ± 0.1 

Distance to lakes  0.0 ± 0.0

Sum of spatial filters  22.5 ± 5.6 

Biodiversity Center, Web‐GIS Atlas of Birds (Bird Banding Survey, Data of recovery records）http://www.biodic.go.jp/birdRinging_en/index.html

Mallard plays important roll to introduce AIV into Japan and maintain AIV in Siberia

Northern Pintail may bring North American lineage of AIV

Risk species

Migration route of the mallard duck Migration route of the northern pintail

Fecal sample is very valuable to get a lot of useful information of wildlife infectious diseases

ConclusionRegarding the surveillance for wildlife diseases,

it is needed;1. To record and report for sharing information of

wildlife diseases internationally2. To understand interface and interaction among live

stocks, wildlife and human3. To understand Ecosystem and Ecology, and also

keep the image of “One Health” in mind

http://iiad.tamu.edu/about-iiad/about/one-health/

Thank you for your attention!

The Sumatran tiger /Harimau (Panthera tigris sumatrae)

Regional Workshop: Training of OIE National Focal Points for Wildlife (4th Cycle) Bali, Indonesia, 4-6 July 2017