Abstract Book of EAVLD2012 - eavld congress 2012

S3 - O - 02
25 YEARS OF PASSIVE SURVEILLANCE OF BATS IN THE NETHERLANDS. MOLECULAR
EPIDEMIOLOGY AND EVOLUTION OF EBLV-1.
Introduction
Since 1986 a reactive surveillance program is running in the
Netherlands to investigate the presence of European Bat
Lyssavirusses (EBLV-1 and EBLV-2) in bats. In this program all
contact cases, with human or pets, are send to the laboratory in
Lelystad and tested for EBLV with a EU prescribed immune
fluorescence test (IFT). Up to date almost 5000 samples have
been tested. The main reservoir for EBLV is Eptesicus serotinus,
334 positive cases so far which is about 22% of the samples
tested, second species is Myotis dasycneme, with almost 4%
positive thus far (Table 1). EBLV-2 has not been detected in the
Netherlands since 1993.
Materials & methods
Bat brains were tested using the prescribed fluorescent antibody
test (FAT). For conformation a real time PCR test on the N-gene
was used. On the same nucleic acid isolation used for the realtime
PCR N and G-gene sequencing was performed on an ABI
sequencer according to the manufacturers protocol. The species
of all submissions were determined by a bat expert.
The MCC phylogenetic tree, estimates of the rate of molecular
evolution (substitutions per site per year) and the TMRCA for the
alignments were inferred using a Bayesian MCMC method in the
BEAST package.
B. Kooi, A. Vogel and E. Van Weezep
Central Veterinary Institute of Wageningen UR, Lelystad, The Netherlands.
Lyssavirus, EBLV, bats, surveillance, molecular epidemiology
Results & Discussion
We performed a molecular epidemiology study on a collection of
more then 40 EBLV-1 viruses that currently circulate in the
Netherlands. Based on the sequences of the coding regions of
the N and G genes a phylogenetic analysis was performed in
which the two distinct lineages or subgroups EBLV-1a and EBLV-
1b were clearly visible (Fig. 1). Surprisingly the larger EBLV-1a
group could be further subdivided into three phylogenetic groups
that were consistent with topology. Two groups were
predominantly found in the northern provinces whereas the third
group was spread from east to west in the middle of the country.
A most recent common ancestor analysis was performed which
confirmed a distinct difference in origin of the two EBLV-1
subgroups. Whereas the EBLV-1a subgroup migrated into
Europe via the east-west route, EBLV-1b apparently entered the
continent from the south, most likely through the Iberian
Peninsula.
.
Table 1: Passive lyssavirus surveillance of bats (1986-2011)
Species tested postitive
no determination 10 -
Myotis mystacinus 22 -
Myotis nattereri 10 -
Myotis daubentonii 124 -
Myotis dasycneme 138 5
Pipistrellus pipistrellus 2216 -
Pipistrellus nathusii 358 -
Nyctalus noctula 77 -
Nyctalus leisleri 4 -
Eptesicus serotinus 1491 334
Vespertilio murinus 13 -
Plecotus auritus 257 -
total 4720 339
Figure 1: Phylogenetic analysis of EBLV-1 based on G-protein
coding regions of EBLV-1 from bats from the Netherlands (2004-
2011)
References
1. Drummond AJ, Rambaut A. BEAST: Bayesian evolutionary analysis by
sampling trees. BMC Evol Biol. 2007;7(1):214.
2. Cliquet, F and Barrat J, 2008. Rabies (chapter 2.1.13). Manual of
diagnostic tests and vaccines for terrestrial animals, OIE (World
Organisation for Animal Health), Paris, 1: 304-322