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FMD Diagnostics
David Paton & Don King
Institute for Animal Health
Institute for Animal Health
Research and surveillance to prevent virus diseases of livestock and their transmission to humans
Institute for Animal Health
• New & improved
diagnostic tests
• Referral diagnosis
• Disease preparedness
and emergencies
• International links
• Training and proficiency
testing
National and International Reference Laboratories
Institute for Animal Health Institute for Animal Health
Talk Outline
• Diagnostic windows
• Suspect case investigation
• Virus characterisation
• Surveillance
• Conclusions
Institute for Animal Health Institute for Animal Health
A
O
Asia1 C
SAT2 SAT3
SAT1
Institute for Animal Health
DAYS
ME
AS
UR
EM
EN
T
14 6 5 4 3 2 1
1 Rapid
confirmation of
clinical signs
2 Active surveillance
for infected animals
(including pre-clinical
cases)
8 7
Diagnostic windows
FMD virus
in blood
Clinical
lesions antibody
response
3 Surveillence for
FMDV exposed
animals
Representative “in contact” cattle data from Alexandersen et al., 2003 and unpublished data from IAH
FMD virus at
mucosal sites
Institute for Animal Health
Relationship between clinical FMD and transmission
Success of reactive strategies, e.g. culling, depends upon how long animals infectious before clinical signs
Proxy measures used based on viraemia/virus excretion
One-to-one contact infection studies suggest shorter interval
Implies early clinical and preferably preclinical diagnosis extremely important and can replace pre-emptive culling
Preclinical screening used in 2007 but currently difficult on large-scale
Charleston et al., Science (2011)
Institute for Animal Health
Clinical diagnosis
July August
9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 1 2 3 4
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July August
11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 1 2 3 4
-15 -14 -13 -12 -11 -10 -9 -8 -7 -6 -5 -4 -3 -2 -1 1 2 3 4 5 6 7 8 9 10
July August
13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 1 2 3 4
-15 -14 -13 -12 -11 -10 -9 -8 -7 -6 -5 -4 -3 -2 -1 1 2 3 4 5 6 7 8
Institute for Animal Health
FMD tests and samples
1. Detection of FMD virus
2. Detection of FMDV-specific antibody (SP/NSP)
• Samples collected: – Tissue (vesicular epithelium)
– Blood (serum from whole blood)
– “probang” samples
– Milk
– Swabs from mucosal surfaces
– Environmental samples (air samples etc..)
Institute for Animal Health
Virus isolation
(BTy or IBRS2)
Ag ELISA
Time to report result (hrs)
1-4 days
~4 hours
Assays for FMDV detection/characterisation
TaqMan®
RT-PCR ~5 hours
1 10 100
Genome
sequencing ~24 hours
Institute for Animal Health
rRT-PCR was widely used to test submitted material, UK 2007
Sample type Number
submitted rRT - PCR
Ag - ELISA
VI
Epithelial suspension
50 50 49 50
Tissue suspension 1 1 1 1
Vesicular fluid 2 1 1 1
Fluid
3 3 - -
Serum 31 15 3086 - 585
EDTA - blood 32
32 - 32
Probang 36 36 - 36
Swab 6 6 1 3
Faecal suspension 1 1 - 1
Total samples tested 3246 3216 52 709
% of samples tested 99.1 1.6 21.8
Increasing reliance upon molecular testing
Reid et al, J Vet Diag Invest 2009
Institute for Animal Health
L 2A 3C Primary cleavages
Poly(C)
L 1A 1B 1C 1D 2A 2B 2C 3A 3B 3C 3D
Secondary cleavages 1B/RNA? 3C 3C 3C 3C 3C 3C
Kilobases
0 1 2 3 4 5 6 7 8
Protease
Carboxy-terminal
self-cleaving Protease Capsid NTP binding* Polymerase
Genome-linked
(VPg)
* putative functions Membrane-binding
VP4 VP2 VP3 VP1 VPG
5’UTR
AAA (n)
3’UTR
variable VP1 region
sequences used for isolate
characterisation
conserved IRES and 3D regions targets for pan-serotype reactive
assays
Genome detection and sequencing
Institute for Animal Health
1000 km
600 mi
© Daniel Dalet / d-maps.com
152
292
6
32
3
135
2
2
2
East Asia incursions: 2010-2011 (O/SEA/Mya-98)
O/CHA/109/2010* (LVRI)
O/CHA/84/2010* (LVRI)
O/CHA/114/2010* (LVRI)
O/CHA/117/2010* (LVRI)
O/CHA/102/2010* (LVRI)
O/SKR/5/2010
O/CHA/57/2010* (LVRI)
O/JPN/2010 (NIAH)
O/RUS/Jul 2010 (ARRIAH)
O/CHA/31/2010* (LVRI)
O/CHA/115/2010* (LVRI)
O/CHA/79/2010* (LVRI)
O/HKN/9/2010
O/HKN/7/2010
O/CHA/51/2010* (LVRI)
O/SKR/4/2010
O/CHA/106/2010* (LVRI)
O/CHA/104/2010* (LVRI)
O/CHA/72/2010* (LVRI)
O/CHA/39/2010* (LVRI)
O/HKN/19/2010
O/CHA/64/2010* (LVRI)
O/HKN/6/2010
O/HKN/1/2010
O/CHA/107/2010* (LVRI)
O/CHA/82/2010* (LVRI)
O/CHA/80/2010* (LVRI)
O/TAI/22/2009 (HQ116269)
O/MYA/5/2009 (HQ116228)
O/MYA/11/2009 (HQ116232)
O/MYA/3/2010
O/MOG/2004 (ARRIAH)
O/HLJOC12/03 (China) (DQ119643)
O/MYA/1/2006
O/MYA/3/2009
O/MYA/2/2006 (HQ116225)
O/VIT/5/2010
O/TAI/20/2009 (HQ116267)
O/MYA/12/2009
O/MYA/3/2008 (HQ116227)
O/LAO/1/2007 (HQ116175)
O/MAY/5/2009 (HQ116213)
O/LAO/2/2008 (HQ116180)
O/TAI/2/2009 (HQ116257)
O/TAI/6/2008 (HQ116253)
O/TAI/2/2008 (HQ116250)
O/TAI/5/2008 (HQ116252)
O/TAI/4/2009 (HQ116259)
O/MYA/2/2008 (HQ116226)
O/TAI/18/2009 (HQ116265)
O/MAY/8/2009 (HQ116216)
O/TAI/3/2009 (HQ116258)
O/LAO/1/2009 (HQ116182)
O/VIT/2/2010
O/TAI/23/2009 (HQ116270)
O/TAI/19/2009 (HQ116266)
O/MAY/20/2009 (HQ116218)
O/TAI/13/2009 (HQ116261)
O/TAI/12/2009 (HQ116260)
O/MOG/66/2010* (ARRIAH)
O/MOG/9/2010
O/MOG/6/2010
O/MOG/77/2010* (ARRIAH)
O/MOG/7/2010
O/MOG/5/2010
O/MOG/CO3/2010* (ARRIAH)
O/RUS/Aug 2010 (ARRIAH)
O/MOG/2/2010
O/MOG/1/2010
O/MOG/56/2010* (ARRIAH)
O/MOG/4/2010
O/MOG/3/2010
O/TAI/189/87* (TRRL)
98
97
73
75
81
86
98
89
86
85
99
99
84
97
99
70
74
84
0.01
Tracking spread of FMDV
Paton et al, Vet Rec 2010
Institute for Animal Health
29-J
ul-
07
19-A
ug
-07
12-A
ug
-07
05-A
ug
-07
26-A
ug
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02-S
ep
-07
09-S
ep
-07
16-S
ep
-07
23-S
ep
-07
Date
IP2b
IP2c
IP3c
IP4b
IP3b
IP5
IP1b(2)
IP1b(1)
IP6b
IP7
22-J
ul-
07
15-J
ul-
07
30-S
ep
-07
IP8
29-J
ul-
07
19-A
ug
-07
12-A
ug
-07
05-A
ug
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26-A
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02-S
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09-S
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16-S
ep
-07
23-S
ep
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Date
IP2b
IP2c
IP3c
IP4b
IP3b
IP5
IP1b(2)
IP1b(1)
IP6b
IP7
22-J
ul-
07
15-J
ul-
07
30-S
ep
-07
IP8
IP2b
IP2c
IP3c
IP4b
IP3b
IP5
IP6b
IP1b(1)
IP1b(2)
IP7
Closest Lab strainIP8
2007 outbreak: real-time forensic tracing
Cottam et al., 2008, PLoS Pathogens
Linking sequence data to infection windows: based on infection dates and lesion aging
Most likely date of infection
(5-7 days prior to clinical signs)
Range of possible incubation
period (14 days)
Clinical disease evident on the
farm
KEY
10 km
X
FMD confirmed
Preclinical (lab only)
No evidence of infection
KEY
5
7
6
8
1b
43b
3c
2b
XX
ALDERSHOT
HEATHROWWINDSOR
XXXX
WOKING
XX
M4
XXGUILDFORD
EGHAM
XX
XXXX
M3
M25
GODALMING
1b
2b
2c
5
4 3c
3b6
7
8
Pirbright
1c
10 km
X
FMD confirmed
Preclinical (lab only)
No evidence of infection
KEY
5
7
6
8
1b
43b
3c
2b
XX
ALDERSHOT
HEATHROWWINDSOR
XXXX
WOKING
XX
M4
XXGUILDFORD
EGHAM
XX
XXXX
M3
M25
GODALMING
1b
2b
2c
5
4 3c
3b6
7
8
Pirbright
1c
10 km
X
FMD confirmed
Preclinical (lab only)
No evidence of infection
KEY
5
7
6
8
1b
43b
3c
2b
XX
ALDERSHOT
HEATHROWWINDSOR
XXXX
WOKING
XX
M4
XXGUILDFORD
EGHAM
XX
XXXX
M3
M25
GODALMING
1b
2b
2c
5
4 3c
3b6
7
8
Pirbright
1c
Institute for Animal Health Institute for Animal Health
IAH2
IAH1
AY593815
IP2b
IP2c
IP3b IP1b(1)
IP1b(2)
MAH
September August
9 substitutions
IAH2
IAH1
AY593815
IP2b
IP2c
IP3c
IP4b
IP3b
IP5
IP6b
IP1b(1)
IP1b(2)
IP7
MAH IP8
UK 2007 Outbreaks
Institute for Animal Health
Next generation
sequencing to understand
FMDV evolution MiSeq (Illumina) C3 C1 C2
Transmission direction
C4
Lost
Drifting
Fixed
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Vaccine efficacy
• Potency evaluation
– With challenge
– Using correlates of protection
• Vaccine strain selection
– Serological methods
– Sequence-based predictions
• Field studies
– Population immunity
– Vaccine effectiveness studies:
subclinical infection
Serologically predicted probabilities of homologous
protection against in vivo outcome of vaccine trials
based on VNT alone (A), and combining VNT and
LPBE (B), showing that combining assays provides
more confidence in protection failures than individual
assays, without affecting ability to predict protection
successes.
Antigenic cartography
Institute for Animal Health
• FMD spreads very rapidly
–Rapid decision required
–Average time to receipt of samples
>24hrs
• Devolved and POC formats
offer potential to significantly
decrease assay time
–Tools to support clinical judgement
• Rapid, simple tests also useful
in labs
Why do we need field tests for FMD?
Institute for Animal Health
Assay Specificity
Assay Sensitivity
New assay formats: considerations
• Reliability – Simple-to-use
– Non-specialist
• Performance – Limit of detection
– Ability to correctly identify infected animals with diverse FMDV strains
• Speed
• Scalability
• Cost ?
Institute for Animal Health
Suspect case of FMD
LOCAL CLINICAL
OBSERVATION
LABORATORY DIAGNOSIS
(Local or NRL)
Field testing vs centralised testing
• Confirmation of 1st case (in a FMD-free country)?
• Strain characterisation • Active surveillance programmes
Field Tests
• FMDV antigen (positive cases) • FMDV na (negative cases)
Diagnostic Tests
Sammin et al Transbound Emerg Dis 2010
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Pen-side diagnostics
Mesosystems: non-invasive air samplers
Infra-red thermography
SVANODIP® FMDV-Ag
Portable molecular assays: Genie III for LAMP and Enigma diagnostics for PCR
Institute for Animal Health
Scatter plot showing hoof temperatures
and their dependence on ambient
temperature.
Differences between rectal and eye
temperature for each animal
29.8 0C
9.8 0C
Gloster et al, BMC Vet Res 2011
Institute for Animal Health
Pre-clinical diagnosis IP2c 2007
• All animals closely
checked for clinical signs
with negative results
• 19 of 58 animals rRT-PCR
positive blood samples
• Indicates near
simultaneous infection of
multiple animals
• First use of real-time
preclinical diagnosis for
FMD in field
Ryan et al Vet Rec 2008
Institute for Animal Health
Active surveillance: use of rRT-PCR for preclinical detection
• Intensively Patrolled Areas (IPA) established
within protection zone (PZ) around Outbreak in
Egham
• Visited every-other day
• Clinical examination
• Blood samples collected and tested for FMDV
by rRT-PCR
• 14 “high risk” cattle herds
• ~ 500 animals
• Reduced un-necessary slaughter
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• Targeted towards structural (SP) or non-
structural proteins of FMDV
• SP assays – separate assays (VNT or ELISA)
required for each serotype
• NSP assays – broadly serotype cross-reactive
(ELISA - 3ABC protein and others)
• IgA and IgM serotype specific ELISAs
Serological assays
Institute for Animal Health
Note that this figure shows the
testing strategy within the
Protection Zone where there
was evidence of recent active
infection. In the Surveillance
Zone, where serosurveillance
commenced at least 21 days
after the last case, it was
considered sufficient to use
the Cedi-NSP alone (i.e.
without VNT) to rule out non-
specific positives found in the
screening Cedi-O ELISA.
Institute for Animal Health
DIVA tests
NSP serology Mucosal IgA
Vaccinated
Vaccine:
Purified Virus
Abs against
Structural proteins
Vaccinated
- infected
NSPs
FMDV
replication
Abs against
Structural and NSPs
Parenteral vaccine
Systemic antibody
Infection
Pharyngeal virus replication
IgA in nasal and
oropharyngeal
secretions
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DIVA Testing with current vaccines
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• Relatively specific detection of infection
• In the past (animals fully recovered)
• Ongoing
• in population - virus circulation
• in individual – virus carrier
• Substantiate freedom from infection
• Lot of testing required
• Specificity challenges
Uttenhthal et al, Exp Rev Vaccine 2010
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DIVA test selection
• NSP serology – Not carrier-specific
– Use in free countries with and without vaccination
– High quality vaccines – less NSP and less viral replication
– Use in endemic situations
– Good commercial kits for 3ABC ELISA
– Multiplex alternatives to Western Blotting (EITB)
• IgA tests – Specific for ongoing virus replication – carriers
– Need to collect nasal fluids
– Not commercially available
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Outbreak preparedness
• Lab capacity – central / devolved / local testing
– biocontainment
– staff
– biorobotics
– kits and reagents
– quality accreditation
• Coordination betweenVS HQ, field and lab – sample submission
– prioritisation
– data management
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Conclusions / Diagnostic gaps
• Increasing use of molecular tests
• Preclinical and field diagnosis mostly not in place
• Data management systems rarely optimal
• Molecular tracing improved but further refinements likely
• Improvements in predicting and monitoring vaccine efficacy
• Decision support needed in relation to vaccine selection
• Commercial kits for SP serology – not yet all serotypes
• Multiplex NSP assays under development
• What to do about carriers after vaccinate-to-live
• Identifying serotypes and incidents of new infection in
complex epidemiological situations
Institute for Animal Health
Institute for Animal Health
Acknowledgements
Institute for Animal Health • Don King
• Satya Parida
• Nigel Ferris
• Nick Knowles
• Simon Gubbins
• Nick Juleff
• Theo Knight-Jones
• Anna Ludi
• Mana Mahapatra
• Bryan Charleston
• Jef Hammond
Principal Collaborators • IZS Brescia
• UK Met Office
• FAO/OIE
• Glasgow University
• Edinburgh University
Principal Funders • BBSRC
• Defra
• European Commission
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