LSD symposium - C. Batten - LSDV diagnostic capabilities at Pirbright

1. LSDV diagnostic capabilities at Pirbright
Dr Carrie Batten, Dr Caroline Wright and Dr Martin Ashby
The Pirbright Institute, United Kingdom

2. Lumpy Skin Disease Symposium; FAO-HQ, Rome; 14-16 March 2023
The Non-Vesicular Reference laboratories at Pirbright

3. Lumpy Skin Disease Symposium; FAO-HQ, Rome; 14-16 March 2023
• 8 WOAH reference laboratories
• Applications to FAO
• Diagnosis/confirmatory testing – 50 samples/year/country/disease
• Technical advice/diagnostic support: ISO/IEC 17025 accreditation
• Validated test protocols
• Lab contingency plan
• Participation (and supply) of PT panels
• Reagent supply
• Research collaborations leading to high impact scientific publications
The Non-Vesicular Reference laboratories at Pirbright

4. Lumpy Skin Disease Symposium; FAO-HQ, Rome; 14-16 March 2023
Primary diagnostic Real-time PCR: Capripox genus
Bowden et al., 2008; Sp: 100%
Good diagnostic sensitivity
Positive at ~ 4dpi (peak viraemia 10-12dpi)
ISO/IEC 17025 Accredited diagnostic tests
ID VET double antigen ELISA: 15-29dpi or vacc
Sp >97.3%, Se:98.6%

5. Lumpy Skin Disease Symposium; FAO-HQ, Rome; 14-16 March 2023
Diagnostic testing regime in the UK setting
APHA Report Case for
Capripox
EDTA
Blood /
Tissue
Serum
Real Time PCR Bowden
et al., 2008
(sample in duplicate)
Real time
PCR result
hours)
Report
Suspicion may be ruled
out at this point if real
time PCR results are
negative
Official veterinarians suspect LSDV
and submit samples to Pirbright
Other parties informed: Chief
Veterinary Officer, competent authority
(Defra), UK policy teams
Prescribed tests are performed in
duplicate
Suggested 2mm
biopsy of lump

6. Lumpy Skin Disease Symposium; FAO-HQ, Rome; 14-16 March 2023
▪ Magnetic silica based extraction MagMax CORE reagents
▪ Performs extraction of 96 sample in <40 mins
▪ 6 KingFishers available for use
KingFisher extraction platform
Rapid front line testing
▪ Capable of fast cycling conditions using fast reagents
▪ Applied Biosystems Path-ID qPCR master mix
▪ 6x QuantStudio 5 Real-Time PCR machines available for use
QuantStudio fast real-time PCR instruments
▪ Analysis duration for diagnostic sample ≤ 2 hours
▪ 12 scientists available for PCR and ELISA testing
▪ 24 hour on-call rota

7. Lumpy Skin Disease Symposium; FAO-HQ, Rome; 14-16 March 2023
Serum neutralisation test (6WP)
Confirmatory serological tests
PRNT – Plaque reduction neutralisation test
Foci (plaque)
formation
Move towards using a GFP expressing strain

8. Lumpy Skin Disease Symposium; FAO-HQ, Rome; 14-16 March 2023
Hong Kong
International disease confirmation
Sample
received
• Samples received 20th November 2020
Real-time
PCR
• Capripox virus confirmed using Bowden et al. assay
Real-time
PCR
• LSDV confirmed using FMCA Lamien et al. assay
Real-time
PCR
• LSDV “vaccine like” strain confirmed using Sprygin et al. assay
Virus
isolation
• Virus isolation successful using MDBK cells

9. Lumpy Skin Disease Symposium; FAO-HQ, Rome; 14-16 March 2023
Distinct areas of CPE leading to clear damage to the cell layer
Progress of infection for attenuated Neethling strain up to 9dpi (rapid)
MDBKs Infected with LSDV
Formation of ‘ridges’ during early stages of infection.
Slower progression
Nigeria 2021 (wild-type)
Control 4 dpi 5 dpi 9 dpi
x10
107 TCID50/ml
x4

10. Lumpy Skin Disease Symposium; FAO-HQ, Rome; 14-16 March 2023
Conventional
PCR
• PCR amplification of RPO30 & GPCR genes
Visualisation
• Amplicons confirmed on agarose gel
Purification
• Amplicons purified using GFX kit
Chain-
termination PCR
• Chain-termination PCR performed using the BigDye Terminator kit
Reading DNA
sequence
• Capillary gel electrophoresis and florescence detection using the ABI 3730 DNA analyzer

11. Lumpy Skin Disease Symposium; FAO-HQ, Rome; 14-16 March 2023
LSDV ‘Cluster’ a
mixture of wildtype
and vaccine strains
Complete RPO30

12. Lumpy Skin Disease Symposium; FAO-HQ, Rome; 14-16 March 2023
Recombinants – effect on diagnostic assays and
ability to differentiate LSDV vaccine v wildtype…
Differentiating Capripoxviruses
Suspected CaPV
sample
CaPV Real time PCR
(Bowden 2008)
Sheeppox Real-time PCR
(Wolff 2021)
Goatpox Real-time PCR
(Wolff 2021)
LSDV field Real-time PCR
(Wolff 2021)
LSDV vaccine-Real time
PCR (Wolff 2021)
If positive
Extract DNA
If negative
Report results
Report result
Report result
Report result
Report result
If positive
If negative
Possible
recombinant strain
If positive
If positive
If positive
alternative approach I

13. Lumpy Skin Disease Symposium; FAO-HQ, Rome; 14-16 March 2023
• Targeted sequencing of alternative capripoxvirus genes
• EEV
• Others
• Whole-genome sequencing…
alternative approach II
Differentiating Capripoxviruses
From virus isolation
Direct from clinical sample using
long-range pre-sequencing PCR

14. Lumpy Skin Disease Symposium; FAO-HQ, Rome; 14-16 March 2023
LSDV training course on behalf of FAO – 2022
LSDV training course at Pirbright 11th – 15th September 2023 – applications open now
https://www.pirbright.ac.uk/training-pirbright/instructor-led-training
LSDV e-learning
https://www.pirbright.ac.uk/training-pirbright/elearning-courses
Confirmatory diagnosis
Research collaborations
International engagement

15. Protecting people, animals, and the environment every day
Drawings: FAO/Chiara Caproni