LSD symposium - A. Haegeman - LSDV transmission by stomoxys stable flies lessons learned from in vivo animal expiriments

1. Andy Haegeman
FAO LSD Symposium 14-16 March 2023, Rome, Italy
LSDV TRANSMISSION BY STOMOXYS
STABLE FLIES:
LESSONS LEARNED FROM IN VIVO
ANIMAL EXPERIMENTS

2. What did we known before….
• Mechanical transmission ticks of LSDV
• Goatpox can be transmitted by Stomoxys

3. Stomoxys and LSDV transmission ?
• Is there live virus present in Stomoxys after feeding?
Overview PCR Overview virus isolation
Dilution factor Nr of Pos. in % average Cp STD Nr of Pos. in %
1/5 5 (5) 100 31,32 0,97 7 (8) 87,5
1/25 5 (5) 100 33,01 0,12 8 (9) 89
1/125 5 (5) 100 36,57 1,29 1 (9) 11
1/625 1 (5) 20 40,3 / ND ND
panCapx RT-PCR and virus isolation results of the vectors following in vitro feeding.
Pure viral stock had a Cp of 22

4. * In vivo feeding transmission model
Stomoxys and LSDV transmission ?
Infection of donors
- intradermal & intravenous
- Infection dose: 107-8 TCID50/animal
-LSDV field strain Israel
Clinical follow Up
Lab-tests (PCR on EDTA blood)
Viremia detected
Field captured Stomoxys are
caged in the desired numbers
and placed on donors for 10
minutes
1hr
of rest
Transfer to
acceptors

5. Stomoxys and LSDV transmission ?
Exposure to a large number of flies / bites (= Mass exposure)
→ Simulates field conditions or stable with limited to no vector control
o Large number of flies per animal / multiple cages / multiple batches
o Multiple in vivo feeding / transfer for several consecutive days
• Trial 1
o 100-200 flies/acceptor for 4 consecutive days
o 4 acceptor animals
20
25
30
35
40
45
50
0 5 6 7 8 9 13 15 16 19 20 23 26 27
qPCR
blood
(CP)
days post infection (dpi)
Donor 1 Donor 3
Donors Acceptors

6. Stomoxys and LSDV transmission ?
Exposure to a large number of flies / bites (= Mass exposure)
→ Simulates field conditions or stable with limited to no vector control
o Large number of flies per animal / multiple cages / multiple batches
o Multiple in vivo feeding / transfer for several consecutive days
• Trial 1
o 100-200 flies/acceptor for 4 consecutive days
o 4 acceptor animals
❖ One acceptor became
▪ Clinical ill (Noduli) → PCR confirmed
▪ Viremic
▪ Seroconverted on IPMA
Conclusion Trial 1
Transmission observed

7. Stomoxys and LSDV transmission ?
• Trial 2
o Batch 1: 100-200 flies/acceptor for 4 consecutive days
o Batch 2: 100-200 flies/acceptor for 4 consecutive days
o 4 acceptor animals
Donors Acceptors
From the first exposure ?? → Long incubation ??
Or from the second exposure ??

8. Stomoxys and LSDV transmission ?
• Trial 2
o Batch 1: 100-200 flies/acceptor for 4 consecutive days
o Batch 2: 100-200 flies/acceptor for 4 consecutive days
o 4 acceptor animals
❖ Two acceptors became
▪ Clinical ill (Nodule) → PCR confirmed
▪ Viremic
▪ Seroconverted on IPMA
Conclusion Trial 2
Transmission confirmed
Confirmed in a similar third trail (1 out 5 acceptors)

9. Stomoxys and LSDV transmission ?
Why transmission in some animals and not in other??
→ Link to:
o Number of flies used per animal ?
o Number of LSDV positive flies per animal?
o Viral load in the vectors?
→ 20 randomly selected S. calcitrans flies per acceptor animal were tested by
real-time PCR

10. Stomoxys and LSDV transmission ?
→ No significant difference in mean Ct values of the flies were found between the group
of viremic and non viremic acceptor animals

11. Stomoxys and LSDV transmission ?
Why do some transmission in some animals ??
→ Link to:
o Number of flies used per animal ? NO
o Number of LSDV positive flies per animal NO
o Viral load in the vectors? NO
Exposure to a large number of flies / bites TRANSMISSION
→ BUT do we need a lot of flies or bites ?????

12. Stomoxys and LSDV transmission ?
Exposure to a limited number of flies / bites (= Minimal exposure)
→ Simulates transport conditions or stable with vector control
o Limited number of flies per animal / one cage (n=20)
o One in vivo feeding / transfer moment
Long incubation period →
cfr Mass exposure trial 2
5 out of 10: fever + nodules

13. Stomoxys and LSDV transmission ?
Question: Can LSDV be transmitted by Stomoxys?
Transmission
lots op vectors YES
Viremic clinical
donors
few vectors YES
BUT what about subclinical infected animals ???

14. Stomoxys and LSDV transmission ?
Subclinical infected animals: What are they ?
Animals which do not develop the typical LSDV nodules but are
PCR positive ( blood, biopsies, organs/tissues at necropsy)
(N=13)
Challenge
Standard protocol
Clinical follow-up
and sampling
Mass exposure
10 min
1 h interval
(N=13)
Acceptors
Donors
Clinical follow-up
and sampling
Trial set-up
(N= 5)

15. Stomoxys and LSDV transmission ?
Two viremic animals:
o Seroconverted (IPMA and ELISA)
o One acceptor developed nodules → clinical disease
o One acceptor did NOT display any clinical sign → subclinical disease !!
o IFNg responsiveness after stimulation of heparin blood

16. Stomoxys and LSDV transmission ?
Question: Can LSDV be transmitted by Stomoxys?
BUT what about subclinical infected animals ???
Conclusion
Transmission from subclinical infected is possible.
Transmission efficiency was similar to that of the clinical infected
animals

17. Stomoxys and LSDV transmission ?
Question: Can LSDV be transmitted by Stomoxys?
YES
• Transmission from clinical animals: low numbers of vectors are
sufficient !!
o Vector control needs to effective to be efficient !!
o Transport can be a risk factor
• Transmission from subclinical animals is possible
o Stamping out of only clinical diseased LSD animals insufficient →
possibly lead to reappearance of LSD
o Vicious cycle: subclinical donor resulted in a subclinical acceptor !!

18. Stomoxys and LSDV transmission ?
This was supported by:

19. Contact
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