FMD Diagnostics - Iowa State University · Detection of FMD virus 2. Detection of FMDV-specific antibody (SP/NSP) ... Clinical disease evident on the farm KEY 10 km X FMD confirmed

FMD Diagnostics

David Paton & Don King

Institute for Animal Health


Research and surveillance to prevent virus diseases of livestock and their transmission to humans


• 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


A

O

Asia1 C

SAT2 SAT3

SAT1


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Representative “in contact” cattle data from Alexandersen et al., 2003 and unpublished data from IAH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


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)


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

-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 11 12

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


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..)


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


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


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


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/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/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


29-J

ul-

07

19-A

ug

-07

12-A

ug

-07

05-A

ug

-07

26-A

ug

-07

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

-07

26-A

ug

-07

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

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



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



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


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


Next generation

sequencing to understand

FMDV evolution MiSeq (Illumina) C3 C1 C2

Transmission direction

C4

Lost

Drifting

Fixed


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


• 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?


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 ?


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


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


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


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


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


• 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


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.


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


DIVA Testing with current vaccines


• 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


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


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


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



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

Documents

FMD Diagnostics - Iowa State University · Detection of FMD virus 2. Detection of FMDV-specific antibody (SP/NSP) ... Clinical disease evident on the farm KEY 10 km X FMD confirmed