The application of genetic markers for The application of genetic markers for European Bat Lyssavirus (EBLV) European Bat Lyssavirus (EBLV)

surveillance in batssurveillance in bats

Sarah HarrisSarah Harris

Rabies and Wildlife Zoonoses Group Rabies and Wildlife Zoonoses Group (VLA – Weybridge, UK)(VLA – Weybridge, UK)

[WHO Collaborating Centre, [WHO Collaborating Centre, Med-Vet-Net]Med-Vet-Net]

Paris, May 2007Paris, May 2007

The application of genetic markers for The application of genetic markers for EBLV surveillance in batsEBLV surveillance in bats

EBLV passive surveillance in UK batsEBLV passive surveillance in UK bats Current methods of bat species ID and problemsCurrent methods of bat species ID and problems Application of genetic markers:Application of genetic markers:

Cytochrome Cytochrome bb gene: cryptic species gene: cryptic species

-actin gene: -actin gene: EBLV-2 virus +ve bat caseEBLV-2 virus +ve bat case Future applications Future applications

EBLV UK passive surveillance (1987-2007)EBLV UK passive surveillance (1987-2007) Bats submitted > 6,700, 16 UK sp, numbers vary Bats submitted > 6,700, 16 UK sp, numbers vary

(Harris (Harris et alet al., 2006)., 2006) 12 bats of 7 non-UK species: e.g. Pond bat (EBLV-2)12 bats of 7 non-UK species: e.g. Pond bat (EBLV-2) UK cases: 5 EBLV-2 virus +ve Daubenton’sUK cases: 5 EBLV-2 virus +ve Daubenton’s Accurate species identification essentialAccurate species identification essential

Morphological characters:Morphological characters: body size, nose-leaf, forearm body size, nose-leaf, forearm

Problems with bat species identificationProblems with bat species identificationTime (~800 bats per year), quality, knowledge, cryptic species Time (~800 bats per year), quality, knowledge, cryptic species

Brandt’s Whiskered

Morphologically Morphologically highly similar:highly similar:

Dentition - wornDentition - wornPenis shape - agePenis shape - age

Forearm - overlapForearm - overlap

Khujand virus identified in Whiskered bat (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

Molecular markers – 2 genesMolecular markers – 2 genes

1.1. CytochromeCytochrome b b –– mtDNA genemtDNA gene for bat phylogeneticsfor bat phylogenetics

Cryptic species Cryptic species

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

Rapid identification of species in suspect bat cases Rapid identification of species in suspect bat cases

So how can we improve rapidity and accuracy So how can we improve rapidity and accuracy of bat species identification?of bat species identification?

Development of Cytochrome Development of Cytochrome bb markers markers

MethodMethod Cytochrome Cytochrome bb (1,200 bp) (1,200 bp) Primers designed (~800 bp), PCR, sequencingPrimers designed (~800 bp), PCR, sequencing Phylogenetic analysis (PAUP: ML analysis, 1000 Phylogenetic analysis (PAUP: ML analysis, 1000

BS)BS) ResultsResults Identified markers for 13 UK species + haplotypes Identified markers for 13 UK species + haplotypes Correctly identified 2 cryptic species Correctly identified 2 cryptic species

Morphological ID was incorrect in some casesMorphological ID was incorrect in some cases

Cytochrome Cytochrome bb phylogenetic analysis: phylogenetic analysis: MyotisMyotis genus genus

Natterer’s bat

Greater Mouse-eared bat

Daubenton’s bat (2 haplotypes)

Bechstein bat

Whiskered (Whiskered (nn = 16) = 16)

Brandt’s (Brandt’s (nn = 12) + 4 ‘Whiskered’ = 12) + 4 ‘Whiskered’

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 Development of -actin -actin markersmarkers

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

(Murray (Murray et alet al., 1990), s., 1990), sequencingequencing Phylip phylogenetic analysis (ML analysis, 100 BS)Phylip phylogenetic analysis (ML analysis, 100 BS)

ResultsResults -actin markers for 11-actin markers for 11 UK species UK species Species ID of EBLV-2 +ve batSpecies ID of EBLV-2 +ve bat

Application of Application of -actin -actin in virus positive casesin virus positive cases September 2004, suspect bat submitted (Surrey, UK)September 2004, suspect bat submitted (Surrey, UK) Standard tests – positive for EBLV-2 Standard tests – positive for EBLV-2 Amplified cDNA generated from RNA (brain), Amplified cDNA generated from RNA (brain), -actin -actin PCRPCR -actin sequence -actin sequence aligned with 32 UK bat sequencesaligned with 32 UK bat sequences 99.85% similarity with Daubenton’s bat99.85% similarity with Daubenton’s bat

603 bps

310 bps

-Actin(348 bps)

M + - 603/04M: marker

+ : ‘+ve’ mouse brain

- : ‘-ve’ control

603/04: M. daubentonii

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

Future applicationsFuture applications EBLV Passive Surveillance systems in EuropeEBLV Passive Surveillance systems in Europe: :

Essential for ID of -ve bat cases as well as +ve Essential for ID of -ve bat cases as well as +ve Up until 2004: >200 EBLV+ve bat cases with species unknownUp until 2004: >200 EBLV+ve bat cases with species unknownMarkers will also enable ID of incomplete/damaged carcasses Markers will also enable ID of incomplete/damaged carcasses

Lyssaviruses in other speciesLyssaviruses in other species

Cytochrome Cytochrome bb gene species ID of LBV-infected Mongoose gene species ID of LBV-infected Mongoose (Markotter (Markotter et alet al., 2006, EID)., 2006, EID)

Disease epidemiology requires accurate informationDisease epidemiology requires accurate information

knownknown host species > host range > epidemiological patterns > host species > host range > epidemiological patterns > potential threat to public/animal healthpotential threat to public/animal health

Acknowledgements Acknowledgements University of Bristol (Prof. Gareth Jones)University of Bristol (Prof. Gareth Jones)

VLA (Dr. Nick Johnson, Dr. Sharon Brookes, Prof. VLA (Dr. Nick Johnson, Dr. Sharon Brookes, Prof. Tony Fooks, and other RWZG members)Tony Fooks, and other RWZG members)

Joint Nature Conservation Committee (JNCC)Joint Nature Conservation Committee (JNCC) DefraDefra Tony HutsonTony Hutson