Bernd Haas

14 March 1958 – 4 October 2015

Replacement of FMDV cattle tongue titration by

in-vitro titration

Aldo Dekker

Introduction

Standardisation FMD challenge tests

●Passage in cattle

●Uniform challenge dose

● OIE manual and European Pharmacopoeia

● 10 000 cattle ID50

● Historically selected

Can we replace tongue titration by in-vitro titration

Titration in cattle tongue: overlap between both cows

Cattle tongue titration: reading at 48 hours

Dataset

27 viruses tested (24 strains)

●A, O, C, Asia-1 and SAT-2

●Most viruses only tested once

●One strain two different passages tested

●A few similar strains tested for different commercial companies

28 experiments

57 cattle used

1197 observations (injection sites)

Titre on primary cells

Min. 1st Qu. Median Mean 3rd Qu. Max.

5.230 7.000 7.530 7.467 7.940 8.440

Virus titre on primary cells

Virus titre (log10PFU/ml)

Fre

qu

en

cy

5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5

05

10

15

20

25

30

Mostly similar titres!

Overall results

Dilution numbermean titre

(log pfu per dose)SD positive / tested

-9 7 -3.0 0 0-8 14 -2.0 0 0-7 28 -0.51 0.67 0.04-6 210 0.54 0.65 0.30-5 378 1.5 0.65 0.50-4 371 2.5 0.72 0.80-3 182 3.4 0.78 0.92-2 7 2.2 0 0.43

Statistical analysis

Logistic regressionFraction positive is

the result variableExplanatory

●Titre injected●Dilution●Virus●Strain●Serotype●animal

-2 0 2 4

0.0

0.2

0.4

0.6

0.8

1.0

Virus titre (PFU log10) injected in the tongue

Fra

ctio

n o

f in

ject

ion

site

s p

osi

tive

Normal logistic regression

Forward regression

Titre injected: First explanatory variable

●Overall 1.3 log10 PFU injected produces a lesion in 50% of the case

Best model: Titre injected + animal

●So significant animal effect

●No strain effect

●Are observations within one animal independent?

Best fitting model: Titre injected + animal

Each animalSame slope Different 50% pointVirus tested not

relevantAverage titre

difference 50% point 0.96 log10 PFU for both cows in one experiment

-2 0 2 4

0.0

0.2

0.4

0.6

0.8

1.0

Virus titre (PFU log10) injected in the tongue

Fra

ctio

n o

f po

sitiv

e in

ject

ion

site

s

Huge difference between cows in same exp.

Cows with 0% or 100% response were removed

difference minimal and maximum titre for each cow in same experiment

Fre

qu

en

cy

0.0 0.5 1.0 1.5 2.0 2.5 3.0

01

23

45

67

Independent observations in one cow?

Observations in one cow are dependent

Model with cow as random variable dilution and original titre are best explanatory variable (is similar to titre injected)

Not possible to detect strain differences as no cattle were injected with two strains

We can replace cattle tongue titration

Huge variation in sensitivity between animals

●Due to variation in animals

●Due to difference in sensitivity of different parts of the tongue

●Due to experimental error

No significant explanation by virus, strain or serotype in the observed results

One study with three vaccines (A, O and C) tested with 10, 10 000 and 1 000 000 bovine ID50 (terré et al. 1972)

●Potency was the same

Relation between cattle ID50 and infection

98% probability for vesicle formation at each injection site using 10 000 ID50

-4 -2 0 2 4

0.0

0.2

0.4

0.6

0.8

1.0

Concentration (log10) cattle ID50 injected in the tongue

Fra

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f in

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ion

site

s o

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po

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Relation between cattle ID50 and infection

Injection at two sites Probability of

infection of cow higher (red line)

90% at 10 ID50

98% at 100 ID50

99.7% at 1000 ID50

99.9% at 10 000 ID50

-4 -2 0 2 4

0.0

0.2

0.4

0.6

0.8

1.0

Concentration (log10) cattle ID50 injected in the tongue

Fra

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f in

ject

ion

site

s o

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Conclusion

Challenge result is not very sensitive to amount of virus

Huge variation in response between cattle

Titration in cattle tongue is not necessary

Proposal: Use 105 TCID50 or PFU for challenge at 2 or more sites.