This document provides guidance on sampling and laboratory diagnosis of foot-and-mouth disease (FMD). It describes the optimal samples to collect from infected animals depending on the stage of infection, such as vesicular epithelium and fluid from animals with lesions. It also outlines appropriate samples from recovered or vaccinated animals like blood. The document reviews the principles of FMD diagnosis using different laboratory tests that can detect virus, viral proteins, or antibodies at various stages. These include virus isolation, antigen and antibody detection tests, and molecular tests like RT-PCR. It provides information on safe packaging and transport of samples to the laboratory for testing.

1. FMD sampling and laboratory
diagnosis
UTC-1
David Paton, Eoin Ryan, Kees Van Maanen,
Nick Lyons, Jenny Maud, Etienne Chevanne

2. Uninfected
Live Virus
by virus isolation
in cell cultures
Viral Proteins by LFD or double
antibody sandwich ELISA
Viral Nucleic Acid
by RT-PCR
Anti-FMD antibodies can be
detected in serum by ELISA or
VNT
FMD Virus infected Recovered (or vaccinated)
Lesion, swab, probang or
clotted blood samples
Virus or viral
components can be
detected
Clotted blood samples
(saliva)
Anti-viral antibodies can
be detected
1-4 days
10-30 minutes (LFD)
4 hours (ELISA)
Around 4 hours 5-18 hours (ELISA)
2-3 days (VNT)
Probang
samples
Principals of FMD Diagnosis

3. 1 Rapid confirmation
of clinical signs
FMD diagnostic windows
2 Pre-clinical surveillance
Virus in blood
Antibody
response
3 Sero-surveillance
for FMDV exposed animals
DAYS
QUANTITY
14654321 87 ● ● ● ●
Source: WRLFMD, Pirbright
Clinical
lesions
virus in
lesions

4. Vesicular epithelium and fluid !! Sample of Choice !!
Always collect if present
The richest source of FMD virus
Highly desired from primary cases
Suitable for all virus detection tests
Blood
Always collect, whether or not vesicles present
Detection of virus in viraemic animals (Clotted or EDTA anticoagulated blood).
Detection of antibodies in recovering/immune animals (Clotted blood).
Oral/nasal swabs
Virus persists longer here than in blood (e.g. 4-5 day old lesions)
Detection of virus by RT-PCR or VI. Also differential diagnosis.
Top Priority Samples

5. Other Samples
Oropharyngeal fluid (probang)
>1 month virus persistence in ~50% infected ruminants (carriers).
Low levels of virus detected by RT-PCR or VI
Cardiac muscle in myocarditis cases
Rich source of virus
Detection by all tests – RT-PCR, Ag ELISA, VI
Milk
Variable amount of virus or antibody
Detection by RT-PCR and by serology
Air or environmental samples
Low levels of often inactivated virus
Detection by RT-PCR.

6. Sampling cases with lesions
Collect: Vesicular epithelium +/- fluid and blood, always!
Heart muscle (myocarditis cases)
Herd-level: Samples from at least 5 animals with obvious lesions should
be sufficient to confirm a diagnosis
– For tissues
At least 2 cm2 of epithelium from unruptured or freshly ruptured vesicles –
fingernail sized amount
Transport medium - equal amounts of glycerine and 0.04 M phosphate
buffer pH 7.2-7.6
– For vesicular fluids (very hard to get!)
Into a plain tube

7. Herd-level: For diagnosis, select at least 10 animals, prioritizing those
with suspicious clinical signs or epidemiological links
-- Fever, depression, lameness, milk drop, healed lesions --
Collect clotted or EDTA blood samples
to obtain serum to detect viraemia or antibodies
Oronasal swabs and/or probangs (ruminants only)
may be of value, laboratory capacity for processing?
Sampling cases with no/old vesicular lesions
Sheep
Calf
Cow

8. Packaging of samples for shipment to the lab
Collect samples in leak-proof, shatter-proof containers and label indelibly;
Disinfect outside of containers, surround with absorbent material and
place in secondary container;
Surround with cool packs and place with submission form in insulated box;
Address outer package and add contents description and hazard warning
Inform laboratory

9. Probang technique
Restrain animal and measure probang against side of head to
calculate distance to throat,
Insert probang over tongue into the oropharynx, avoiding teeth,
Move back and fourth 4-6 times to collect fluid from the soft
palate area,
Withdraw the probang cup with the head held up. Discard the
sample if it contains rumen material (pH=5).
Cattle: tip the fluid into a tube containing 2-3 ml of neutral pH
0.08M PBS.
Sheep/goats: insert probang head into a wide-necked tube
containing 2-3 ml of neutral pH 0.08M PBS and agitate.
Wash probang thoroughly between animals: 3 bucket system
WATER
4% NA2CO3
or 0.2% CITRIC ACID
WATER

10. Field diagnosis of FMD (pen-side)
Lateral-flow devices
(LFDs)
Sensitivity ~80%
Rapid confirmation
of positives
Able to confirm
negatives
T-COR 8 PCR machine in action
(real-time amplification of the target DNA
is displayed on the screen)
Molecular assays
(RT-PCR, LAMP)
Sensitivity>95%

11. Sample preparation for Ag-LFD

12. SVANODIP® FMDV-Ag Test: Test Procedure
Reading results after 10 minutes
No line in the ”T” position
indicates a NEGATIVE test result
A line in the
”C” position
indicates a
valid test
A line in the ”T” position
indicates a POSITIVE test result
After field testing,
the LFD can be sent
to the lab for RT-PCR

13. Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Absorbent
pad
Release
pad Blue-coloured latex beads
coated with FMDV-specific Ab
Clinical sample
Containing FMDV
FLOW FLOW
Rapid (<10 mins) confirmation of FMD in the field (simple, stable, robust, no need of cold chain,
suited for field conditions)
Recognises all 7 FMDV serotypes
Similar assay performance (sensitivity) to lab-based Ag-ELISA
Only for vesicular epithelium or fluid
Lateral Flow Device (LFD)

14. • Investigating suspect cases of FMD and quantifying
the prevalence of FMDV infection
• Substantiating freedom from infection at population
or individual animal level (often for trade)
• Evaluating population immunity (post vaccination
monitoring) and vaccine matching
FMD Serology: applications

15. FMD Serology: SP/NSP tests and the DIVA principle
NSPs
Infection with
live virus
SP antibodies
- Serotype-specific
- Correlate to
protection
NSP antibodies
- Pan-serotype reactive
- Used for DIVA testing
Viral non-structural
proteins (NSP)
Inactivated purified
virus (structural
proteins, SP)
Live virus
KEY TO FIGUREVaccination with
purified vaccine
Vaccinated
- infected
NSPs
Vaccine virus
preparation
Purification

16. FMD serological tests detect antibodies directed at
either:
• FMD viral structural proteins (SP) that are variable and make
up the capsid or shell of virus particles e.g. SP ELISAs or VNT
or
• FMD viral non-structural proteins (NSP) that are conserved
and involved in intracellular virus replication e.g. NSP ELISAs
Types of FMD Serology

17. O A C Asia 1 SAT 1 SAT 2 SAT 3
anti-SP Antibodies serotype-specific
Seven different assays, one for each FMD virus type
anti-NSP antibodies identical for 7 serotypes
O / A / C / Asia 1 / SAT 1 / SAT 2 / SAT 3
NSP tests  A unique assay for all FMD virus types

18. Both can be used to detect animals that have been infected
with FMDV but their markedly different specificities will influence
the choice of which one to use in particular situations
• Only an NSP test can
– detect antibodies to any FMDV serotype
– differentiate antibodies due to infection from those due to
vaccination
• Only an SP test can
– measure protective immunity
– identify the serotype (more difficult in endemic settings)
Differences in Use of SP and NSP tests

19. What samples would you take?
FMD suspect case in Gharbaya,
Egypt, March 2012
What samples should I have
taken?
What results would you
expect?

20. Thanks for your attention
Questions on sampling and diagnostic?