Bernd Haas 14 March 1958 – 4 October 2015. Replacement of FMDV cattle tongue titration by in-vitro titration Aldo Dekker.

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Presentation transcript:

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 ● cattle ID 50 ● 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

Cattle tongue titration: result example

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.MedianMean3rd Qu.Max

Virus titre on primary cells Mostly similar titres!

Overall results

Statistical analysis  Logistic regression  Fraction positive is the result variable  Explanatory ● Titre injected ● Dilution ● Virus ● Strain ● Serotype ● animal

Normal logistic regression  Forward regression  Titre injected: First explanatory variable ● Overall 1.3 log 10 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 animal  Same slope  Different 50% point  Virus tested not relevant  Average titre difference 50% point 0.96 log 10 PFU for both cows in one experiment

Huge difference between cows in same exp.  Cows with 0% or 100% response were removed

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, and bovine ID 50 (terré et al. 1972) ● Potency was the same

Relation between cattle ID 50 and infection  98% probability for vesicle formation at each injection site using ID 50

Relation between cattle ID 50 and infection  Injection at two sites  Probability of infection of cow higher (red line)  90% at 10 ID 50  98% at 100 ID 50  99.7% at 1000 ID 50  99.9% at ID 50

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 10 5 TCID 50 or PFU for challenge at 2 or more sites.