Transcript (Vulpes vulpes) to European bat lyssaviruses types-1 and

2nd International Conference of Rabies in Europe, 2007
Experimental Infection of Foxes with
European Bat Lyssaviruses
type-1 and 2
E. Picard1, S.M. Brookes2, D.M. Healy2, J. Barrat1, L. Johnson2 , C.
Patron1, E. Litaize1, M. Biarnais1,
F. Cliquet1 & A.R. Fooks2
1 WHO Collaborating Centre for Research and Management in Zoonoses Control, OIE Reference Laboratory for
Rabies, Community Reference Laboratory for Rabies Serology, AFSSA Malzeville, France
2 WHO Collaborating Centre for the Characterization of Rabies and Related Rabies Viruses, Rabies and Wildlife
Zoonoses Group, Veterinary Laboratories Agency, Weybridge, United Kingdom
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2nd International Conference of Rabies in Europe, 2007
State of art (1) :
What about the situation of bat rabies in Europe?
• Bats are important Lyssavirus reservoirs in Europe,
reported since 1954
– infected with EBLV-1 and EBLV-2
• 800 cases of bat rabies detected in Europe
• Distribution of bat rabies in all european countries
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2nd International Conference of Rabies in Europe, 2007
State of art (2) :
What about the transmission of Bat Rabies (EBLV-1 and 2) to
terrestrial (wild and domestic) mammals?
• Few cases of « spillover »
– four fatal infections reported in man (and 3 nonconfirmed cases) since 1977
– cross the species barrier:
• sheep in 1998 and 2002 in Denmark
• stone marten in 2001 in Germany
• antibodies from a cat in Denmark (2004)
• So far, no spill over of EBLV-2 into terrestrial
mammals has been reported
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2nd International Conference of Rabies in Europe, 2007
Objectives of the study
• Assess the susceptibility of wild animals (silver foxes
Vulpes vulpes) to the infection of EBLV type 1 and 2
• Investigate wether or not foxes, main reservoir and vector
of rabies in Europe, may transmit virus (EBLV-1 or
EBLV-2)
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2nd International Conference of Rabies in Europe, 2007
Material
– 40 animals used in the study
• Captive silver foxes
• Individual cage ; each animal identified with micro-chip
• General behaviour daily recorded
– 26 IM inoculated, 6 IC inoculated, 8 controls
– Sampling
• Saliva (after inoculation)
– Once a week during two months, then once a month for 2/3 years
• Brain samples and sub-maxillary glands
» Collected on day of euthanasia or death
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2nd International Conference of Rabies in Europe, 2007
Methods used to establish rabies diagnosis (2)
• Rabies diagnosis :
– Fluorescent antibody test
– Cell culture inoculation test
« detection of antigen and infectious particles »
• Molecular tools : RNA extraction and RT-PCR
« detection of viral nucleoprotein RNA » (606 bp of N gene)
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2nd International Conference of Rabies in Europe, 2007
Experimental design- IC and IM challenges
group
Infected group
5
1
3.5
IM
Control group
5
1
n.a.
n.a.
EBLV-2
Infected group
3
2
2.8 ; 3.2
IC
EBLV-1
Infected group
3
2
4.4 ; 4.7
IC
Control group
1
2
n.a.
n.a.
Infected group
6
3
2.7 ; 3.7 ; 4.4 ; 4.7
IM
Control group
1
3
Infected group
15
4
Control group
1
4
EBLV-2
EBLV-1
EBLV-1
n.a. not administered
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Inoculated Dose
(Log MIC LD50)
number
of foxes
Injected virus
n.a.
3.0 ; 3.7 ; 4.4
n.a
route
n.a.
IM
n.a.
2nd International Conference of Rabies in Europe, 2007
Pathogenicity, EBLV-1 and 2, IC infections results
Infected group
(N°2)
salivary
excretion**
route
number
IC
2
4.7
2/2
Yes (2 & 6 days)
1
4.4
1/1
Yes (2 days)
2
2/2
1
3.2
2.8
Yes (3 & 5 days)
Yes (n.c.)
Neg (0/3)
1
/
no
Neg (0/1)
Infected group
(N°2)
IC
Control group
(N°2)
n.a.
n.a. not administered
n.c. not recorded
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clinical
signs
Log
MIC LD50
dead
1/1
0/1
1*
D12 and D22
D282
3* D8 & D10
4* D10
2*
Rabies diagnosis
(FAT)
Pos (2/2)3*
Neg (0/3)
Pos (1/1)4*
Pos (2/2)1*
Pos (1/1)2*
Neg (0/1)
2nd International Conference of Rabies in Europe, 2007
Pathogenecity IC trial results and conclusions (1)
• All infected foxes IC with EBLV-1 and 2
– showed clinical signs of rabies
– EBLV-1 foxes died 8 and 10 days p.i.
– EBLV-2 foxes died 12-282 days p.i
• Rabies diagnosis
– Viral antigen, infectious particles and viral RNA in brain (6/6).
– Salivary glands positive (3/6)
• No salivary excretion by using cell culture inoculation and
no specific RNA detection using RT-PCR.
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2nd International Conference of Rabies in Europe, 2007
Pathogenicity, EBLV-1 and 2, IM infections results
route
Infected group
(N°3 & 4)
IM
IM
number
dead
1
1
4.7
4.4
0/1
0/1
no
no
2
2
3.7
2.7
1/2
0/2
Yes (2 days)
no
5
4.4
0/5
no
5
3.7
0/5
no
5
3.0
2/5
salivary
excretion
Neg (0/6)
Neg (0/15)
3.5
0/5
no
Neg (0/5)
Control group
(N°1 & 2)
n.a.
7
/
0/7
no
Neg (0/7)
D14
D17 & D24
Neg (0/5)
Neg (0/1)
Pos (2/5)2*
Yes (2 & 4 days)
5
2*
Neg (0/1)
Neg (0/1)
Neg (0/2)
IM
1*
rabies diagnosis
(FAT)
Pos (1/2)1*
Infected group
(N°1)
n.a. not administered
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clinical
signs
Log
MIC LD50
Neg (0/5)
Neg (0/7)
2nd International Conference of Rabies in Europe, 2007
Pathogenecity IM trial results and conclusions (2)
• IM trials performed with EBLV-1 and EBLV-2 :
– Death of 3 foxes (out of 7 infected animals) challenged with 3 and
3.7 log MIC LD50 of EBLV-1 (minimal doses) ; presence of rabies
clinical signs
– None of foxes challenged with high dose of EBLV-1 died
– None of foxes challenged with EBLV-2 dead p.i (5/5).
• No salivary excretion by using cell culture inoculation and
no specific RNA detection using RT-PCR.
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2nd International Conference of Rabies in Europe, 2007
Conclusion
• Low susceptibility of foxes to EBLV type 1 and 2
• Animal-animal spillover of EBLVs is unlikely
– because the virus is rapidly cleared from the
host suggesting that a bat variant of rabies virus
(i.e. EBLV) is strongly host-adapted.
• The lack of viral RNA detection in saliva from the
I.C. challenged foxes suggests that fox-to-fox
transmission may also be unlikely.
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2nd International Conference of Rabies in Europe, 2007
Acknowledgments
• The work was supported by :
– the Direction Générale de l’Alimentation (French Ministry of Agriculture)
– The Department for Environment Food and Rural Affairs (Defra)
• From the French team we would like to thank :
– Josiane Ambert, Jacqueline Bailly, Michel Munier, Amélie Verdot, Marine
Wasniewski and the "serological team" for their excellent technical
support. Patricia Grosgeorge, Audrey Hamen, Sébastien Kempff, Anouck
Labadie, Jean Luc Schereffer and Laetitia Tribout and Marie José
Duchène for the bibliography.
• From the UK team we would like to thank :
– members of the Rabies and Wildlife Zoonoses group plus Verity Crossan,
and Anne Adernomu for PCRs, and Dan Hicks from the Histopathology
group.
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