Yamakawa ta ds in japan (fftc workshop in manila)201607

Important Transboundary Animal Diseases
in Japan
FMD
-Current Status and Research Activities -
Makoto Yamakawa
National Institute of Animal Health (NIAH)
National Agriculture and Food Research Organization (NARO)
JAPAN
Workshop on Management and Control of Important Transboundary Animal Diseases in the Asian Pacific Region
Manila, Philippines, July 25-29, 2016

Recent outbreaks of important transboundary
animal diseases in Japan (Since 2000)
Foot-and-mouth disease (FMD)*
2000, 2010
Highly pathogenic avian influenza (HPAI)*
2004, 2007, 2010-2011, 2014-2015
Porcine epidemic diarrhea (PED)
Nationwide outbreak was observed in 2013-2014.
Arthropod-borne viral diseases in cattle
Akabane and related Bunyaviral diseases, Bovine ephemeral fever and so on
*OIE free status: FMD (February 2011), HPAI (April 2015)
These were successfully eradicated by culling infected animals and poultry
according to the basic policy on disease control measures!

Foot-and-mouth Disease (FMD)
One of the most important infectious diseases for livestock industry
・ FMD virus can infect many kinds of cloven-hoofed animals, such as
cattle, pigs, sheep and goats and is highly contagious.
・ FMD virus causes acute febrile disease with characteristic vesicular
lesions in mouth, nose, mamma and foot.
・ 7 distinct serotypes (O, A, C, Asia1, SAT1, SAT2, SAT3) are identified.
・ FMD causes economic damage by nutrition disorder and dyskinesia.
・ FMD is a huge obstacle to the international trade of animals and animal
products.

History of FMD Outbreaks in Japan
•FMD occurred only twice in 100 years
•Good animal quarantine system
•Geographical advantage as islands
Year
Animal
species
Place
(prefectures)
No. of slaughtered
animals
Serotype/topotype of
the virus
1900-1908 cattle 18 prefectures 4,051 unknown
2000 cattle
Miyazaki
Hokkaido
740
O/ME-SA
topotype
PanAsia lineage
2010
cattle
pigs
goats
sheep
Miyazaki
297,808
(including vaccinated
animals)
O/SEA topotype
Mya-98 lineage
Effective to protect incursion
of FMD
Pathogenicity and infectivity of the 2000 strains seemed to be weaker than those
of the 2010 strain.

Outbreak of FMD in 2010
<2010 Outbreak > Type O
20 April to 4 July
Miyazaki Prefecture
292 farms, 211,608 animals
Cattle: 37,454
Swine: 174,132
Goat: 14
Sheep: 8
In this area, cattle
and pig farms are
clustered close
together.

1
2
1 1 1
2
1 1
2
4 4
5 5
8
3 3
7
5
9
7
8
10
4
10
11
7 7 7
8
6
3
2
7
6
9
5
6
4
1
2
1 1 1 1
2 2
1 1 1 1
1
1
1 1
1
2
4
8
4
1
2
3
1
2
3
1
3
7
2
4
3 1
3
4
1
1
3
1
1
1
5
1
1
1
2
3
1
292 outbreaks
(including 1 goat
farm)
0
50
100
150
200
250
300
350
0
2
4
6
8
10
12
14
16
【1例】4/20
【1例】4/22
【０例】4/24
【0例】4/26
【3例】4/28
【1例】4/30
【2例】5/2
【2例】5/4
【12例】5/6
【6例】5/8
【11例】5/10
【5例】5/12
【5例】5/14
【10例】5/16
【12例】5/18
【13例】5/20
【10例】5/22
【7例】5/24
【9例】5/26
【3例】5/28
【6例】5/30
【6例】6/1
【5例】6/3
【3例】6/5
【1例】6/7
【1例】6/9
【2例】6/11
【1例】6/13
【0例】6/15
【0例】6/17
【0例】6/19
【0例】6/21
【0例】6/23
【0例】6/25
【0例】6/27
【0例】6/29
【0例】7/1
【0例】7/3
牛発生農場数豚発生農場数
（戸）（戸）
No. of Outbreaks
per day
Time course of the number of outbreaks in 2010
(Cattle and Pigs) Total No. of
Outbreaks
No. of Outbreaks
in cattle
No. of Outbreaks
in pigs
Date
Pigs amplify the virus and excrete huge amount of infectious virus particles.

Number of animals queuing to be slaughtered
Control Measures adopted
by government:
Stamping out
Movement restriction
Emergent vaccination
(Vaccine to kill)
20/April/2011 19/May 4/June
Occurrence
of 1st case
Vaccination
decided
Act on special measures
for FMD came into force
27/July
All restriction
lifted
Recovery of OIE
Status
FMD-free country
where vaccination
is not practiced
5/February/2011

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40km
FMD Vaccination
in Japan
First experience for us!
• All cloven-hoofed animals in the
movement restriction zone (10 km
radius)
• Vaccine containing 6 PD50 of serotype
O, Manisa strain (inactivated and
purified viruses with oil adjuvant )
• r1 value was estimated at 0.35 by
vaccine-matching test
• Destruction of the vaccinated animals
(vaccination to kill)
Cattle 45,950
Pigs 79,606
Others 118
Total 125,556
Number of vaccinated animals
1,066 farms

Epidemiological findings in 2010
FMD virus was supposed to be introduced from FMD affected countries
in Asia (probably via people or goods) from the result of genetic analysis
of 2010 isolates. But incursion route was unknown (We could not identify).
Retrospective surveillance indicated that FMD virus has already invaded
in the middle of March, 2010 (almost 1 month before outbreak).
The delayed confirmation of the first case was one of the factors for the
epidemics.
Main route of FMD transmission among farms was considered to be the
movement of farm workers and livestock transport vehicles. The local
spread was presumed in highly affected area.

FMD control activities
Prevention
to avoid introduction
Update the latest disease information in Asia and the world
Timely alert the outbreaks to farmers, travelers and local veterinary authorities
Impose strict control at ports (points of entry) by Animal Quarantine Services, MAFF
Enhance biosecurity measures at farms (Annual reporting, on site inspection…)
for early detection and notification
When typical or suspected cases are found at farms, livestock owners should notify
the livestock hygiene service centers (prefectural governments). Clinical samples are
collected and sent to National Institute of Animal Health (NIAH) for laboratory diagnosis
according to the FMD control guidelines.
Diagnosis
All laboratory tests are conducted at Exotic Disease Research Station, NIAH in Tokyo
(OIE collaborating center for diagnosis and control of animal diseases).
Containment: FMD-positive animals and animals kept in the same farm are destroyed.
Stamping out, disinfection, movement restriction, investigation of the surrounding farms
In the movement restriction zone, animals showing typical symptoms are recognized and destroyed immediately
as FMD cases based on the photographs of lesions.
Dairy
management is
important.

FMD control activities
Compensations by MAFF: for rapid and effective control
Losses incurred by stamping out and precautionary culling (100%)
Costs for incineration and burial of livestock corpses and contaminated goods
(50% in case of stamping out and 100% for precautionary culling)
Sales reduction (50%) caused by movement restrictions
Preparedness
Securement of human resources and materials necessary for emergent response
Application of ‘Map system for controlling animal diseases’ established in 2012
Regular simulation exercises :
All prefectural governments carry out desk-top simulation exercises organized by MAFF.
Many prefectures also conduct demonstration-type exercises.
Regular update of national FMD control guidelines
Reservation of the vaccine for emergent use (Type O, A, Asia1): annually reviewed
Establishment of International cooperation
Sharing disease information through international meetings
(OIE/FAO GF-TADs, OIE/JTF FMD Scientific Meeting, Southeast Asia & China FMD Meeting (SEACFMD)
Symposium on Prevention and Control of Foot and Mouth Disease in East Asia……and so on)
Technical transfer to Asian countries
(OIE Twinning project :Japan-Mongolia from 2016)

2000 (September)
Regained the free status without vaccination
2010 (April-July) FMD Outbreak
2011 (February)
Regained the free status without vaccination
2000 (March-May) FMD Outbreak
FMD Status of Japan
 FMD Free country without vaccination for long time until 2000
Stamping out
Stamping out + Emergent vaccination(vaccine to kill)

Research activities on FMD
in National Institute of Animal Health, JAPAN
Exotic Disease Research Station, NIAH in Tokyo.
BSL 3 bio-containment laboratory for FMD diagnosis
and FMD research works
We have finally started FMD research after 2000 epidemic in Japan.
Kyushu Research Station
(Kagoshima)
NIAH, headquarters
(Tsukuba)
Hokkaido Research Station
(Sapporo)
Exotic Diseases Research Station
(Kodaira, Tokyo)

RT-PCR
Virus Isolation
negative
positive
Antigen detection ELISA
Isolated
virus
Neg
Cont.O
A
C
SAT1
SAT2
SAT3
Asia1
SVD
Strong Weak
Cont.
RT-PCR
Antigen detection
ELISA
Virus isolation
2.5-6 hr: most rapid and sensitive
4 - 6 hour: simple and quick, less sensitive
than PCR
2～7days: time-consuming but
important
Consuming time for diagnosis
Laboratory Diagnoses of FMD in Japan
Antibody Detection
(in outbreaks and
surveillances)
•LPB ELISA
•NT (in necessary)
We have to focus our studies on
improvement and development of
diagnostic methods.

Newly developed ELISA for FMDV-antigen detection
We have developed antigen-detection ELISA method using
monoclonal antibodies against FMDV serotypes O, A, C and Asia1.
ELISA developed at Exotic
Diseases Research Station is
more sensitive than the
international standard method
(indirect sandwich ELISA), for
detection of FMDV and
identification of its serotype.
We have inspected its utility at World Reference Laboratory of FMD (Pirbright Lab. in UK).
We will use this ELISA together with the international standard method for diagnosis of FMD.
J. Clinical Microbiology 47(11) 3663-3668, (2009). PLOS ONE, 9(4) e94143,（2014）.
Multi serotypes detection
Serotype-specific
detection
Multi-serotypes Serotype specific (O) indirect sandwich
Positive rate (%): sample
(No. of ELISA+ / No. of PCR+)
Positive rate (%): farm
(No. of ELISA+ / No. of PCR+)
57
(102/178)
85
(66/78)
64
(114/178)
87
(68/78)
9
(15/176)
14
(11/78)

We have made an anti-FMDV monoclonal antibody reacting with all serotypes and seven serotype-
specific monoclonal antibodies. We are now trying to develop an immune-chromatography for detecting
viral antigens and for identifying serotypes simply and rapidly in collaboration with private companies.
Development of a simple diagnostic method that can be used in the field
to detect viral antigens
Immune-chromatography kit for detecting viral antigen (final version):
a case of detection of SAT2
control
➞
Anti-SAT1➞
Anti-Asia1➞
Anti-C
➞
Anti-A
➞
Anti-O
➞
Monoclonal antibodies
against each serotype of
FMDV are applied on the
strip.
①FMDV particles in the
clinical sample bind
Gold colloid labelled
anti-FMDV monoclonal
antibody.
③FMDV particles are
captured by detecting
monoclonal antibody on
the strip. A pink line
appears.
② FMDV particles
move in the strip by
capillary action Anti-IgG antibody is applied
Anti-SAT2
➞
Anti-SAT3
➞
➞
Anti-FMDV
PLoS ONE, 2015 10(8): e0134931
Antigen Detection and Serotyping
Lateral Flow Antigen Detection System
for FMDV

We established rapid and sensitive virus-isolation system. A fetal goat tongue cell line （ZZ-R 127）and a
swine kidney cell line expressing αvβ6 integrin (FMDV receptors) （LFBK-αvβ6） were introduced from
Germany and USA, respectively. We have investigated their sensitivity to FMDV using specimens from
the experimentally infected cattle, goats and pigs.
Rapid isolation of FMDV from clinical samples using highly
sensitive cell lines
Animal No. of samples*
Rate of isolation (%)
ZZ-R 127 IB-RS-2
Cattle
Goat
Pig
80
77
78
78.8
63.6
42.3
37.5
23.4
20.5
Total 235 61.7 27.2
*RT-PCR positive samples
Comparison of isolation of FMDV from infected animals
*Sera, saliva, nasal discharge, feces and OP fluids from experimentally infected animals
Animal No. of samples*
Rate of isolation (%)
ZZ-R 127 LFBK αvβ6
Cattle
Pig
210
144
5.7 (12/210)
18.8 (27/144)
7.1 (15/210)
18.8 (27/144)
Total 354 11.0 (39/354) 11.9 (42/354)
Both cell lines are highly sensitive
and will be used for isolation of
FMDV together with cell lines
described in OIE manual of
diagnostic tests.
Journal of Veterinary Diagnostic Investigation, 25, 770-774 (2013)
Journal of Veterinary Diagnostic Investigation, 27, 516-521 (2015)

Genetic analysis of the Japanese strains of FMD virus (type O)
isolated in 2010 outbreak
・ We have isolated many strains of FMD virus from clinical samples in the 2010 epidemic in
Japan.
・ The L-fragment genes were amplified by RT-PCR and sequenced by using a next generation
sequencer.
・ Nucleotide sequences of 2010 isolates showed more than 99.5% identity to the sequence of
initial isolate obtained from the first case of the epidemic without any genetic deletion or insertion.
・These results indicated that a single strain of FMD virus was introduced from overseas and
its nucleotide sequence has changed gradually during the epidemic.
0
50
100
150
200
250
300
350
0
2
4
6
8
10
12
14
16
【1例】4/20
【０例】4/24
【3例】4/28
【2例】5/2
【12例】5/6
【11例】5/10
【5例】5/14
【12例】5/18
【10例】5/22
【9例】5/26
【6例】5/30
【5例】6/3
【1例】6/7
【2例】6/11
【0例】6/15
【0例】6/19
【0例】6/23
【0例】6/27
【0例】7/1
cattle
pig
No.ofcases
Total No. of cases
Date of outbreak
(2010 in Miyazaki Prefecture)
Virus isolation was conducted using samples collected
from April to June, 2010
Phylogenetic analysis of L-fragment of
FMDV (type O)
Japanese
strains
isolated in
2010

1. Sharing disease and scientific information
2. Early notification of the FMD outbreak to OIE Member
countries in the region and OIE headquarters
3. Strengthen the border control to prevent FMD virus entry
4. Promotion of collaborative projects among FMD laboratories in
the region
5. Technical supports to South East Asian countries for diagnosis
of FMD
6. Financial supports to provide FMD vaccines to South East
Asian countries
To reduce FMD outbreaks in the Asian region

Thank you for your attention !

Yamakawa ta ds in japan (fftc workshop in manila)201607

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