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The application of genetic markers for European Bat Lyssavirus (EBLV) surveillance in bats. Sarah Harris Rabies and Wildlife Zoonoses Group (VLA – Weybridge, UK) [WHO Collaborating Centre, Med-Vet-Net] Paris, May 2007. The application of genetic markers for EBLV surveillance in bats.

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the application of genetic markers for european bat lyssavirus eblv surveillance in bats
The application of genetic markers for European Bat Lyssavirus (EBLV) surveillance in bats

Sarah Harris

Rabies and Wildlife Zoonoses Group

(VLA – Weybridge, UK)

[WHO Collaborating Centre,

Med-Vet-Net]

Paris, May 2007

the application of genetic markers for eblv surveillance in bats
The application of genetic markers for EBLV surveillance in bats
  • EBLV passive surveillance in UK bats
  • Current methods of bat species ID and problems
  • Application of genetic markers:

Cytochrome b gene: cryptic species

b-actin gene: EBLV-2 virus +ve bat case

  • Future applications
eblv uk passive surveillance 1987 2007
EBLV UK passive surveillance (1987-2007)
  • Bats submitted > 6,700, 16 UK sp, numbers vary

(Harris et al., 2006)

  • 12 bats of 7 non-UK species: e.g. Pond bat (EBLV-2)
  • UK cases: 5 EBLV-2 virus +ve Daubenton’s
  • Accurate species identification essential

Morphological characters:body size, nose-leaf, forearm

problems with bat species identification

Time (~800 bats per year), quality, knowledge, cryptic species

Problems with bat species identification

Morphologically

highly similar:

Dentition - worn

Penis shape - age

Forearm - overlap

Brandt’s Whiskered

Khujand virus identified in Whiskeredbat (Tajikstan, Kuzmin et al., 2003)

Older cases in unusual species:

P. Pipistrellus/P. nathusii: EBLV-1 Germany (1985-1992)

N. noctula: EBLV-1 (1991) EBLV-2 (1985) Ukraine

Canine isolate (1955) Former Yugoslavia – species not present

slide5

So how can we improve rapidity and accuracy

of bat species identification?

Molecular markers – 2 genes

1. Cytochrome b –mtDNA genefor bat phylogenetics

Cryptic species

2. b-actin – housekeeping gene for EBLV PCR at VLA

Rapid identification of species in suspect bat cases

development of cytochrome b markers
Development of Cytochrome b markers

Method

  • Cytochrome b (1,200 bp)
  • Primers designed (~800 bp), PCR, sequencing
  • Phylogenetic analysis (PAUP: ML analysis, 1000 BS)

Results

  • Identified markers for 13 UK species + haplotypes
  • Correctly identified 2 cryptic species

Morphological ID was incorrect in some cases

cytochrome b phylogenetic analysis myotis genus

Natterer’s bat

Greater Mouse-eared bat

Daubenton’s bat (2 haplotypes)

Bechstein bat

Whiskered (n = 16)

Brandt’s (n = 12) + 4 ‘Whiskered’

Cytochrome b phylogenetic analysis: Myotis genus

20% of morphological ID Whiskered bat’s were genetically ID as Brandt’s bats

Current UK pop estimates: 40,000 and 30,000 – accurate for conservation?

development of b actin markers
Development of b-actin markers

Method

  • RNA extracted from brain sample of bats
  • PCR (primers ~313 bp) b-act1 / b-act2

(Murray et al., 1990), sequencing

  • Phylip phylogenetic analysis (ML analysis, 100 BS)

Results

  • b-actin markers for 11 UK species
  • Species ID of EBLV-2 +ve bat
application of b actin in virus positive cases
Application of b-actin in virus positive cases
  • September 2004, suspect bat submitted (Surrey, UK)
  • Standard tests – positive for EBLV-2
  • Amplified cDNA generated from RNA (brain), b-actin PCR
  • b-actin sequence aligned with 32 UK bat sequences
  • 99.85% similarity with Daubenton’s bat

348 bpb-Actin:M. daubentonii EBLV-2 +ve bat

M + - 603/04

M: marker

+ : ‘+ve’ mouse brain

- : ‘-ve’ control

603/04: M. daubentonii

603 bps

b-Actin

(348 bps)

310 bps

future applications
Future applications
  • EBLV Passive Surveillance systems in Europe:

Essential for ID of -ve bat cases as well as +ve

Up until 2004: >200 EBLV+ve bat cases with species unknown

Markers will also enable ID of incomplete/damaged carcasses

  • Lyssaviruses in other species

Cytochrome b gene species ID of LBV-infected Mongoose

(Markotter et al., 2006, EID)

  • Disease epidemiology requires accurate information

known host species > host range > epidemiological patterns >

potential threat to public/animal health

acknowledgements
Acknowledgements
  • University of Bristol (Prof. Gareth Jones)
  • VLA (Dr. Nick Johnson, Dr. Sharon Brookes, Prof. Tony Fooks, and other RWZG members)
  • Joint Nature Conservation Committee (JNCC)
  • Defra
  • Tony Hutson