1. FMD outbreak investigation procedures – the importance of lesion aging for selection of animals for sampling and for establishing timeline of events

2. Acknowledgments: Nick Juleff, IAH (Pirbright) and Naci Bulut (Turkey)

3. First Phase of support to African FMD labs
objective to identify FMDV serotypes circulating in northern Sudan, borders of Ethiopia, somali ecosystem of Kenya
overall –achieved the objective in
however:
donor (EC) indicates future support must achieve a higher level of virus submission/typing than in Phase I (more efficient)
limited information associated with the source outbreaks – were they “”sporadic cases”” or part of a major epidemic passing through the area?
when “emergent FMDV” are found, who could help assess the risk? role of the lab network?

4. Frequent problems reported during the first Phase of support to African labs
sampling “border regions”” - difficulty of vet teams to reach herds with suspected outbreaks
no virus isolated from animals sampled (lesions sampled too late? low virus present + loss in transport)
virus detected by rtRT-PCR at WRL Pirbright but not isolated (low virus present + loss in transport?)
low level of isolation from probang samples (where used)
limited value to serology in recovered animals (does not allow VI, genetic or antigenic analysis)

5. Widespread problem?
problem: samples collected too late (> 5-6 days cut-off?)
solutions:
train vets to search for and sample only acute lesions (<4 days)
problem: what to do if only recovered animals found?
train vets to continue searching until find young lesions?
train vets to collect serum and other samples to increase chance of determining serotype
greater use of probang sampling?
more use of swab sampling for rtRT-PCR?

6. Diagnostic windows: intense but short time window of virus in lesions
2 Active surveillance for infected animals
(including pre-clinical cases)
1 Rapid confirmation of clinical signs
3 sero-surveillence for FMDV exposed animals
antibody response
Clinical
lesions
FMD virus in blood
MEASUREMENT
Virus in lesions 1 2 3 4 5 6 7 8
● ● ● ● 14
DAYS
Representative “in contact” cattle data from Alexandersen et al., 2003 and unpublished data from IAH

7. Recovered (or vaccinated)
Principals of FMD Diagnosis
Recovered (or vaccinated)
Uninfected
FMD Virus infected
Probang samples
Lesion, swab, probang or clotted blood samples
Clotted blood samples
(saliva)
Virus or viral components can be detected
Anti-viral antibodies can be detected
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
1-4 days
10-30 minutes (LFD)
4 hours (ELISA)
Within a day
5-18 hours (ELISA)
2-3 days (VNT)

8. FMDV diagnosis: Window of detection by different techniques/tissues: note difficulty after 4 days!!
clinical signs of FMD
Epithelium
Serum
Milk
Cell culture
/ Ag ELISA
RT-PCR
Epithelium
Serum
Milk
ELISA Structural
ELISA Non structural
VNT
Antibodies

9. Example – immediate confirmation FMD in lesions < 4 days (LFD= penside test) ETC2 course June 2009
Lesion age incorrect? epithelium hard to collect
at 8 days and do not expect to be Ag-positive

10. Why estimate age of lesion?
Age range of lesions-particularly the oldest lesion
Essential for case history and epidemiological report
Pre-requisite to determine origin of infection
Duration and weight of virus excretion
Prediction of further spread 10

11. Establishing Timelines
Estimate age of oldest lesion on farm
date lesions first appeared (NB: error margin)
Subtract incubation period 1-14 days  window for introduction of virus
most likely incubation period of 2-5 days before lesions appeared
therefore direct questioning to events that occurred 2-5 days before lesions (animal contacts, movements in)
participative methods may help resolve disputes about events
where hiding or shame affects willingness to answer
in endemic areas, FMD may occur every year ...or more often
participative information gathering helps to understand why , when, how...
This slide shows the estimated infection window (blue; dark blue is the most likely period of 2-5 days before disease first appeared) and period when clinical disease was present (orange) at each IP during the UK 2007 outbreak. On the date of diagnosis, the age of the oldest lesion was estimated, and the orange and blue windows calculated accordingly.
Overlaid is a black line interrupted with white circles; this “tree” represents the genetic evolution of the viruses found on each IP. It can be seen that the phylogeny of the viruses matches up with the timelines provided by the lesion age estimates.
The combination of lesion age estimated and novel virus sequencing techniqued provide a powerful epidemiological tool. 11

12. Risk periods NB: virus can be excreted before clinical signs appear
Time of contact is very important
Tracings onto farm: incubation period 1-14 days before lesions appear; most likely 2-5 days
Tracings off farm: refer to virus excretion period
NB: virus can be excreted before clinical signs appear
Milk: can contain virus 4 days before disease appears 12

13. Estimating the age of lesions
Photographs of contact exposure – field and experimental
Clinical manifestation may vary between strains – especially sheep
Complicated by secondary infections
Between day 0 and 5 – one day margin of accuracy 13

14. Estimating the age of lesions
State Veterinary Journal, 5,Number 3, October 1995, pages 4 – 8 14

15. Cattle - Day 1
One day old vesicle, ruptured when tongue drawn from mouth 15

16. Cattle - Day 2
Note raw epithelium, clear edge to lesion and no deposition of fibrin 16

17. Cattle - Day 3
Lesions start to lose their sharp demarcation, fibrin deposition starts to occur on edge of lesions 17

18. Cattle - Day 4
Considerable fibrin deposition has occurred and regrowth of epithelium is evident at edge of lesion 18

19. Cattle - Day 5
Note progressive loss of lesion margination and extensive fibrin infilling 19

20. Cattle - Day 7
Field example. Extensive scar tissue formation and healing has occurred. Some fibrin deposition is usually still present 20

21. Sheep - Day 2
Note sharp lesion margins 21

22. Objectives of the Clinical investigation
to confirm the presence of clinical signs of FMD
to collect suitable samples to confirm FMD infection has occurred
search for fresh/most recent cases, less than 5-6 days age !
to identify the oldest lesions in the unit, to estimate the timing of entry of infection
search for the oldest lesions! use serology if animals recovered and lesions healed

23. Sampling from lesions
Lesion material is the richest source of FMDV and the sample of choice for diagnosis
Samples from ~ 4 animals with obvious (and early) lesions should be sufficient to confirm a diagnosis
The most suitable materials are
Vesicular epithelium, vesicular fluid, heart muscle from myocarditis cases
For tissues
At least 2 cm2 of epithelium from unruptured or freshly ruptured vesicles
Transport medium - equal amounts of glycerine and 0.04 M phosphate buffer pH
For vesicular fluids
Plain, small volume tube

24. Virological Samples Urgent Hazardous (unless inactivated – for PCR)
send as soon as possible, by most direct route
always give advance warning to lab and ETA
correct external package label to identify urgency
do not package together with other samples of less urgency
Hazardous (unless inactivated – for PCR)
package and label properly (UN/IATA dangerous goods standards)
Fragile
keep cool but not frozen, except by prior arrangement, if long delay
avoid extremes of pH therefore use buffered media
Adequate quantities
Separate tube for each animal
Correct forms

25. Sampling in absence of lesions – incubation period or recovered
For diagnosis select ~ 6 animals, prioritizing those with suspicious clinical signs
Fever, depression, lameness, hot feet
Collect clotted blood samples to obtain serum to detect viraemia
Recovery period:
clotted blood samples for antibodies
Oronasal swabs and/or probangs may be of value, but need to take account of laboratory capacity for processing (VI and PCR based methods)

26. Time needed for current assays for FMDV detection
Virus
isolation
(CTY or IBRS2)
1-4 days
~4 hours
Ag ELISA
Automated
TaqMan®
RT-PCR
~5 hours 1 10
100
Time to report result (hrs)

27. ‘Pen-side’ test for antigen detection
Lateral Flow Device (LFD)
Not serotype specific
Based on technology used in pregnancy test kits
Similar sensitivity to Ag-ELISA
High specificity
Used to test epithelium or vesicular fluid
Result within minutes
Used on-farm in UK 2007
Used in regional lab in Turkey in 2009
Relatively low cost per test

28. Probang samples 3 bucket “system” Different sizes:
Aliquot 2-3 ml 0.08 M PBS with 0.01% BSA, 0.002% phenol red and antibiotics, adjusted to pH 7.2 per animal to be sampled.
Cattle: pour probang sample from the cup into a wide-necked bottle & examine visually for quality. Add 2ml, including visible cellular material, to equal volume of buffer and mix. The final pH of a normal sample should be ~pH 7.6.
Sheep: insert probang cup directly into a disposable container into which has been dispensed 3 ml of buffer solution and gently mix
Samples taken from some animals may be heavily contaminated with ruminal contents - these should be discarded and the animal's mouth should be flushed with water before repeat sampling
3 bucket “system”
Water
4% Na2CO3 or 0.2% citric acid
Different sizes:
Sheep
Calf
Cow

29. How do we improve efficiency of FMDV typing in Eastern African situations?
the answers should come from this workshop!
inevitably:
we need field vets trained to correctly select and sample animals
we need more local typing services
we need rapid typing for emergent strains (<7 days)
we need suitable serotype specific penside tests
we will move towards preserving RNA at sample site (safety, preserves samples)
we may send RNA rather than virus to ref labs (but not yet)

30. Thank you Nick Juleff, Pirbright Eoin Ryan, Ireland Naci Bulut, Turkey
all the EuFMD training team (Nadia Rumich, Enrique Anton)