Goats and sheep as sentinels for tick-borne encephalitis (TBE) virus - Epidemiological studies in areas endemic and non-endemic for TBE virus in Germany

Abstract

The aim of the study was to examine grazing goats and sheep as specific sentinels for characterization of the tick-borne encephalitis virus (TBEV)-related risk in an area by means of serosurveillance tests in the German federal states Baden-Wuerttemberg, Bavaria, Thuringia, North Rhine-Westphalia, Lower Saxony, Schleswig-Holstein, and Mecklenburg-West Pomerania. A total of 3590 sheep sera and 3793 goat sera was collected in 2003 and 2006-2009 and were examined by ELISA screening and confirmed by serum neutralization test. Considerable differences in seroprevalence were seen between single flocks in districts in Baden-Wuerttemberg, Bavaria, and Thuringia with values between 0 and 43% which confirmed the patchy pattern of TBEV foci that can range in size from very small to large. The here described serological screening may be a helpful tool for an early warning system of a potential TBEV risk. Testing of 1700 ticks by real-time RT-PCR in two districts in Baden-Wuerttemberg revealed only one positive tick, thus illustrating the problems of expensive and time-consuming tick collection.

Full text

Ticks
and
Tick-borne
Diseases
3 (2012) 27–
37
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and
Tick-borne
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Original
article
Goats
and
sheep
as
sentinels
for
tick-borne
encephalitis
(TBE)
virus
–
Epidemiological
studies
in
areas
endemic
and
non-endemic
for
TBE
virus
in
Germany
Christine
Klausa,∗,
Martin
Beera,
Regine
Saierb,
Ute
Schauc,
Udo
Moogc,
Bernd
Hoffmanna,
Roland
Dillera,
Jochen
Süssa
aFriedrich-Loeffler-Institut,
Jena,
Greifswald-Insel
Riems,
Germany
bInstitute
of
Food
Science
and
Biotechnology,
University
of
Hohenheim,
Stuttgart,
Germany
cThüringer
Tierseuchenkasse,
Animal
Health
Service,
Jena,
Germany
a
r
t
i
c
l
e
i
n
f
o
Article
history:
Received
17
June
2011
Received
in
revised
form
22
August
2011
Accepted
19
September
2011
Keywords:
Tick-borne
encephalitis
virus
Seroprevalence
Sheep
Goats
Natural
TBEV
foci
Sentinels
a
b
s
t
r
a
c
t
The
aim
of
the
study
was
to
examine
grazing
goats
and
sheep
as
specific
sentinels
for
characterization
of
the
tick-borne
encephalitis
virus
(TBEV)-related
risk
in
an
area
by
means
of
serosurveillance
tests
in
the
German
federal
states
Baden-Wuerttemberg,
Bavaria,
Thuringia,
North
Rhine-Westphalia,
Lower
Saxony,
Schleswig-Holstein,
and
Mecklenburg-West
Pomerania.
A
total
of
3590
sheep
sera
and
3793
goat
sera
was
collected
in
2003
and
2006–2009
and
were
examined
by
ELISA
screening
and
confirmed
by
serum
neutralization
test.
Considerable
differences
in
seroprevalence
were
seen
between
single
flocks
in
dis-
tricts
in
Baden-Wuerttemberg,
Bavaria,
and
Thuringia
with
values
between
0
and
43%
which
confirmed
the
patchy
pattern
of
TBEV
foci
that
can
range
in
size
from
very
small
to
large.
The
here
described
sero-
logical
screening
may
be
a
helpful
tool
for
an
early
warning
system
of
a
potential
TBEV
risk.
Testing
of
1700
ticks
by
real-time
RT-PCR
in
two
districts
in
Baden-Wuerttemberg
revealed
only
one
positive
tick,
thus
illustrating
the
problems
of
expensive
and
time-consuming
tick
collection.
© 2011 Elsevier GmbH. All rights reserved.
Introduction
Tick-borne
encephalitis
(TBE)
is
the
most
important
viral
tick-
borne
zoonosis
in
Europe
(Süss
et
al.,
2008b)
and
occurs
also
in
some
parts
of
Asia
(Süss,
2011).
TBE
virus
(TBEV)
circulates
between
ticks
and
hosts
in
geographically
strictly
limited
natural
foci
(endemic
areas)
whose
size
can
range
from
large
to
very
small.
This
is
in
contrast
to
Borrelia
burgdorferi
s.l.,
the
most
important
bacterial
tick-borne
zoonosis
that
is
ubiquitously
endemic
in
all
areas
where
Ixodes
ricinus,
its
main
vector,
occurs
in
central
Europe.
However,
the
reason
for
the
patchy
pattern
of
TBEV
is
not
well
understood
up
to
now.
The
spatial
and
temporal
spread
of
natural
TBE
foci
has
been
observed
by
several
research
groups
and
can
be
influenced
by
many
different
factors
such
as
virus
prevalence,
vector
occurrence,
host
activity,
socio-economic
as
well
as
climate
and
microclimate
changes
(Randolph
and
Rogers,
2000;
Kˇ
riˇ
z
et
al.,
2004;
Danielová
et
al.,
2008;
Randolph,
2008;
Randolph
et
al.,
2008;
Süss
et
al.,
2008b;
Holzmann
et
al.,
2009;
Korenberg,
2009).
It
has
also
been
∗Corresponding
author
at:
Friedrich-Loeffler-Institut,
Institute
of
Bacterial
Infec-
tions
and
Zoonoses,
Naumburger
Str.
96a,
D-07743
Jena,
Germany.
Tel.:
+49
3641
804
2231;
fax:
+49
3641
804
2228.
E-mail
address:
christine.klaus@fli.bund.de
(C.
Klaus).
observed
that
a
natural
TBE
focus
detected
in
the
1960s
(Süss
et
al.,
1992)
now
has
become
extinct
(Klaus
et
al.,
2010a).
In
order
to
iden-
tify
the
reasons
for
the
patchy
pattern
of
TBE
foci,
it
is
important
to
gather
more
information
on
the
eco-epidemiological
situation
and
the
TBEV
circulation
in
an
area,
and
to
collect
and
examine
all
available
data.
In
Germany,
TBE
in
humans
is
a
notifiable
disease,
and
autochthonous
human
cases
are
evaluated
and
registered
by
the
Robert
Koch
Institute
(Robert
Koch
Institute,
2011).
Risk
areas
(districts)
are
defined
as
areas
with
a
mean
of
at
least
1
case
per
100,000
inhabitants
in
the
course
of
5
years
(Robert
Koch
Institute,
2007).
In
the
future
however,
increasing
vaccination
rates
may
lead
to
misinterpretions
of
the
TBE
risk
in
a
given
area,
and
it
is
recom-
mended
to
gather
additional
information
either
by
investigation
of
wild
animals
such
as
mice
(Achazi
et
al.,
2011)
or
by
detec-
tion
of
TBEV
antibodies
in
free-ranging
animals.
TBEV
prevalence
in
ticks
can
provide
this
additional
information.
The
well-established
method
based
on
the
collection
and
analysis
of
ticks
is,
however,
time-consuming,
expensive,
and
often
not
very
effective.
Due
to
the
low
prevalence
of
TBEV
in
ticks
(Oehme
et
al.,
2002;
Süss
et
al.,
2004;
Gäumann
et
al.,
2010)
or
in
wild
animals
such
as
mice
(Achazi
et
al.,
2011),
a
huge
number
of
samples
is
needed.
In
veterinary
medicine,
clinical
cases
of
TBE
are
seldom,
but
they
have
been
described
in
dogs
(Leschnik
et
al.,
2002),
horses
(Waldvogel
et
al.,
1981;
Grabner,
1993),
and
monkeys
1877-959X/$
–
see
front
matter ©
2011 Elsevier GmbH. All rights reserved.
doi:10.1016/j.ttbdis.2011.09.011

28 C.
Klaus
et
al.
/
Ticks
and
Tick-borne
Diseases
3 (2012) 27–
37
Table
1
Seroprevalence
of
TBEV-specific
antibodies
in
sheep
collected
in
2008
and
2009
in
Baden-Wuerttemberg.
District
Flock
(village)
No.
of
sera
No.
of
positive
sera
Seroprev.a(%)
Bodenseekreis
(1)
BS1
(Bermatingen)
20
0
0
BS2
Salem) 100 9
9
BS3
(Salem) 30
12
40
BS4
(Überlingen)
20
3
15
Different
4
1
Bodenseekreis
(1),
total
174
25
14.4
Esslingen
(2) 20 0
Ludwigsburg
(3) 10 0
Ravensburg
(4) 44
0
0
Reutlingen
(5)
94
0
0
Schwäbisch-Hall
(6)
13
0
0
Sigmaringen
(7)
20
0
0
Tübingen
(8) 49
0
0
Tuttlingen
(9) 2
0
0
Districts
(1)–(9)
total
399
25
6.3
aSignificant
difference
between:
flock
BS1
and
BS3
(p
=
0.002);
flock
BS3
and
BS4
(p
=
0.007).
(Süss
et
al.,
2007,
2008a).
Large
domestic
animals
such
as
goats,
sheep,
and
cattle
are
potential
hosts
for
I.
ricinus
and
develop
an
antibody
titer
after
infection
with
TBEV
without
showing
specific
clinical
signs
of
illness
(Gresiková
and ˇ
Reháˇ
cek,
1959;
Gresiková
et
al.,
1975;
Gresiková
and
Calisher,
1988).
These
animals
are
viraemic
over
a
very
short
time
period
only
(Van
Tongeren,
1955),
but
sero-convert
for
a
longer
time
period.
Therefore,
it
is
possible
that
large
domestic
animals
grazing
on
meadows
nearly
all
year
round
with
a
potentially
close
contact
to
ticks
could
serve
as
sen-
tinels
and
could
be
used
for
calculating
the
TBE
risk
in
a
given
area.
Sera
of
goats
and
sheep
are
easy
to
collect;
it
is
also
possible
to
use
sera
collected
for
other
routine
diagnostic
test
in
the
flock.
Sheep
and
goats
are
optimal
sentinels
because
they
are
rather
sedentary
and
stay
on
the
meadows
owned
or
leased
by
the
flock
owner
or
on
meadows
used
on
agreed
terms
of
a
contract,
e.g.
for
rural
conservation.
A
first
clue
was
provided
by
the
examination
of
a
small
goat
flock
in
a
TBE
non-risk
area
in
Thuringia
where
some
single
human
TBE
cases
had
occurred.
Interestingly,
a
TBEV-specific
antibody
titer
was
detectable
in
the
serum
of
one
goat
for
more
than
1
year
(Klaus
et
al.,
2010c).
Therefore,
the
aim
of
this
study
was
the
serological
investigation
of
grazing
animals
(goats,
sheep)
to
analyze
their
use
as
specific
sentinels
for
characterization
of
the
TBE
risk
in
a
given
area.
Materials
and
methods
In
2003
and
from
2006
to
2009
(mainly
in
2009),
3793
goat
sera
and
3590
sheep
sera
were
collected
and
examined
for
TBEV
anti-
bodies
by
ELISA
and
confirmed
by
serum
neutralization
test
(SNT).
The
sera
were
collected
in
several
districts
in
Baden-Wuerttemberg,
Bavaria,
North
Rhine-Westphalia,
Thuringia,
Lower
Saxony,
Schleswig-Holstein,
and
Mecklenburg-West
Pomerania.
According
to
the
definition
of
the
Robert
Koch
Institute
(Robert
Koch
Institute,
2007),
TBE
risk
areas
have
been
in
Baden-Wuerttemberg,
Bavaria
and
Thuringia.
North
Rhine-Westphalia,
Lower
Saxony,
Schleswig-
Holstein,
and
Mecklenburg-West
Pomerania
so
far
have
no
TBE
risk
areas,
however
single
autochthonous
human
TBE
cases
have
been
registered.
For
this
study,
these
districts
were
divided
into
TBE
risk
areas
and
TBE
non-risk
areas
at
the
time
of
sera
collection
(2003
for
the
sera
from
Bavaria
collected
in
2003,
2009
for
all
other
sera).
TBEV
antibodies
were
determined
by
ELISA
using
the
IMMUNOZYM
FSME
IgM
kit
(Progen
Biotechnik
GmbH,
Heidelberg,
Germany)
according
to
the
manufacturer’s
instructions,
but
in
a
modified
version
without
the
blocking
step
for
IgG
to
determine
the
whole
Ig-fraction
(Müller,
1997).
This
is
an
all-species
kit,
and
Müller
(1997)
validated
the
kit
for
dog
sera.
In
dogs,
less
than
5
units/l
were
defined
to
be
negative,
5–7
units/l
borderline,
and
a
score
of
8
and
more
units/l
was
defined
to
be
positive.
Labora-
tory
tests
with
defined
goat
and
sheep
sera
revealed
that
this
kit
was
also
suitable
for
detecting
TBEV
antibodies
in
small
ruminants
(Klaus
et
al.,
2010b,
2010c).
In
goats
and
sheep,
less
than
5
units/l
were
defined
to
be
negative,
5–14
units/l
borderline,
and
a
score
of
more
than
14
units/l
was
defined
to
be
positive.
In
order
to
avoid
false-positive
results
all
ELISA-positive
sera
were
confirmed
by
SNT
as
gold
standard
according
to
Holzmann
et
al.
(1996),
but
in
a
mod-
ified
version
(Klaus
et
al.,
2010c),
and
only
sera
confirmed
by
SNT
were
further
considered
in
these
investigations.
In
2
districts
of
Baden-Wuerttemberg
which
had
been
defined
as
TBE
risk
areas
for
a
longer
time
(districts
Emmendingen
and
Ortenaukreis),
1700
ticks
were
collected
in
2008,
sorted
and
tested
for
TBEV
by
2
independent
real-time
RT-PCR
assays,
one
according
to
Schwaiger
and
Cassinotti
(2003)
in
a
modified
ver-
sion
(Klaus
et
al.,
2010a),
the
other
one
according
to
Klaus
et
al.
(2010b).
Collected
ticks
were
ground
up
in
a
mixer
mill
with
3
stainless
steel
beads
of
3
mm
(Retsch
GmbH,
Haan,
Germany)
and
400
␮l
medium
(MEM
Earle,
Biochrom
AG,
Berlin,
Germany).
Aliquots
of
these
single-tick
suspensions
were
pooled
(50
␮l
from
10
male
or
female
adults
or
10
nymphs),
and
140
␮l
of
each
pool
were
used
for
RNA
extraction
according
to
the
manufacturer’s
instructions
using
the
QIAamp
Viral
RNA
Mini
Kit
(Qiagen
GmbH,
Hilden,
Germany).
From
TBEV-positive
pools,
each
tick
was
retested
individually.
As
a
statistical
method,
the
Mann–Whitney-Test
in
SPSS
Version
17.0
(significant
difference:
p
≤
0.05)
was
used
to
compare
sero-
prevalences
of
samples
from
different
flocks
in
the
same
district,
between
districts,
and
between
TBE
risk
and
TBE
non-risk
areas
(as
it
was
possible
due
to
the
number
of
collected
sera).
Results
In
Baden-Württemberg,
399
sheep
sera
were
collected
in
2008
and
2009,
and
2240
goat
sera
were
collected
from
2006
to
2009.
Sera
were
only
collected
in
districts
located
in
defined
TBE
risk
areas.
The
seroprevalence
observed
in
sheep
was
6.3%
and
in
goats
4.6%
(Tables
1
and
2).
In
2
districts,
where
1700
ticks
were
col-
lected
and
tested
for
TBEV-RNA,
only
one
female
tick
was
found
to
be
positive
(Table
3).
In
Bavaria
in
2003,
676
sheep
sera
were
collected
in
districts
defined
as
TBE
risk
areas
at
that
time,
and
793
sera
were
collected

C.
Klaus
et
al.
/
Ticks
and
Tick-borne
Diseases
3 (2012) 27–
37 29
Table
2
Seroprevalence
of
TBEV-specific
antibodies
in
goats
collected
in
2006–2009
in
Baden-Wuerttemberg.
District
Flock
(village)
No.
of
sera
No.
of
positive
sera
Seroprev.a(%)
Alb-Donau-Kreis
(1)
5
0
0
Biberach
(2) 10 0
Bodenseekreis
(3) 4
0
0
Breisgau-HS
(4)
B1
(Feldberg)
47
0
0
B2
(Heuweiler)
18
3
17
B3
(Horben)
191
11
5.8
B4
(Löffingen)
24
0
0
B5
(Münstertal) 22 2 9
B6
(Münstertal) 20 0
0
B7
(Münstertal) 21
0
0
B8
(Münstertal)
29
0
0
B9
(Münstertal)
42
0
0
B10
(Münstertal)
35
0
0
B11
(Sulzburg)
28
12
43
Different 236
4
1.7
Breisgau-HS
(4),
total
713
32
4.5
Emmendingen
(5)
E1
(Emmendingen) 25 5 20
E2
(Freiamt)
21
0
0
E3
(Freiamt)
24
0
0
E4
(Freiamt)
27
1
4
E5
(Glottertal)
80
0
0
E6
(Glottertal) 95 0 0
E7
(Mundingen)
98
2
2
Different 84
0
0
Emmendingen
(5),
total
454
8
1.8
Esslingen
(6)
16
1
6
Freiburg
(7)
33
0
0
Konstanz
(8) 81 13
Lörrach
(9)
243
2
0.8
Ortenaukreis
(10)
O1
(Schuttertal)
93
10
11
O2
(Schuttertal)
98
33
34
O3
(Schuttertal)
103
0
0
O4
(Oberharmersbach) 23 7 30
Different
38
1
3
Ortenaukreis
(10),
total
355
51
14.4
Ostalbkreis
(11)
7
0
0
Ravensburg
(12)
29
0
0
Sigmaringen
(13)
20
0
0
Tübingen
(14) 17 0 0
Tuttlingen
(15)
197
1
0.5
Waldshut
(16)
110
0
0
Zollernalbkreis
(17) Z1
(Balingen)
27
6
22
Single
sample
1
0
0
Zollernalbkreis
(17),
total
28
6
21.4
Baden-Wuerttemberg
(1–17),
total
2240
102
4.6
aSignificant
difference
between:
districts
Breisgau/HS
and
Emmendingen
(p
=
0.008);
districts
Breisgau/HS
and
Ortenaukreis
(p
=
0.000);
districts
Breisgau/HS
and
Zoller-
nalbkreis
(p
=
0.001);
districts
Emmendingen
and
Ortenaukreis
(p
=
0.000);
districts
Emmendingen
and
Zollernalbkreis
(p
=
0.000).
in
districts
defined
as
TBE
non-risk
areas
at
that
time.
In
2008
and
2009,
759
sheep
sera
were
collected,
740
in
districts
defined
as
TBE
risk
areas
in
2009
and
19
in
one
TBE
non-risk
district.
The
sero-
prevalence
in
sheep
was
3.8%
in
TBE
risk
areas
and
2.8%
in
TBE
non-risk
areas
in
2003,
and
10.7%
in
TBE
risk
areas
in
2008/9.
Only
19
sera
were
examined
from
one
district
defined
as
TBE
non-risk
area
in
2008/9,
and
no
positive
sera
were
found
(Tables
4
and
5).
In
Bavaria,
goat
sera
were
collected
as
well
in
2003
and
2009,
275
Table
3
Ticks
collected
in
two
districts
in
Baden-Wuerttemberg.
District
Females
Males
Nymphs
Larvae
Total
Positivea
Emmendingen
26
44
358
98
526
0
Ortenaukreis 81
86
1007
0
1174
1
female
Total 107
130
1365
98
1700
1
aTested
with
two
different
real-time
RT-PCR
(Schwaiger
and
Cassinotti,
2003;
Klaus
et
al.,
2010b).

30 C.
Klaus
et
al.
/
Ticks
and
Tick-borne
Diseases
3 (2012) 27–
37
Table
4
Seroprevalence
of
TBEV-specific
antibodies
in
sheep
collected
in
Bavaria
in
2003.
District
Flock
No.
of
sera
No.
of
positive
sera
Seroprev.a(%)
2003
TBE
risk
areas
Altötting
(1) 50
0
Amberg-Sulzbach
(2) AS1
122
10
8.2
AS2
26
1
4
Amberg-Sulzbach
(2),
total
148
11
7.4
Ansbach
(3)
71
0
0
Bamberg
(4)
8
0
0
Bayreuth
(5) 29 0 0
Deggendorf
(6) 4
0
0
Erlangen-Höchstadt
(7)
26
1
4
Forchheim
(8)
61
1
2
Freyung-Grafenau
(9)
29
0
0
Kronach
(10)
17
0
0
Landshut
(11) L1
64
6
9
Mühldorf
(12) 33
0
0
Neuburg-Schrobenhausen
(13)
2
0
0
Neumarkt
Oberpfalz
(14) 111 4 3.6
Neustadt
an
der
Waldnaab
(15)
26
1
4
Passau
(16)
3
0
0
Rosenheim
(17)
R1
20
2
10
Roth
(18)
11
0
0
Schwandorf
(19)
5
0
0
Straubing-Bogen
(20) 30 0
Total
(1–20)
676
26
3.8
2003
TBE
non-risk
areas
Amberg
(1)
14
0
0
Bad
Kissingen
(2)
39
1
3
Bad
Tölz-Wolfratshausen
(3)
33
0
0
Dachau
(4) 156 0 0
Ebersberg
(5)
30
0
0
Eichstätt
(6)
12
1
8
Erding
(7)
24
0
0
Erlangen
(8)
12
1
8
Freising
(9) 48 1 2
Garmisch-Partenkirchen
(10)
132
2
1.5
Landsberg
am
Lech
(11) 7
1
14
Miesbach
(12)
16
0
0
München
(13)
57
1
2
Neu-Ulm
(14)
10
0
0
Oberallgäu
(15)
13
0
0
Schweinfurt
(16)
S1
39
6
15
Starnberg
(17) 10 0
0
Weilheim-Schongau
(18)
11
0
0
Würzburg
(19)
29
1
3
Wunsiedel
im
Fichtelgebirge
(20) W1
71
6
8
W2
21
1
5
Different
9
0
0
Wunsiedel
im
Fichtelgebirge
(20),
total
101
7
6.9
Total
(1–20)
793
22
2.8
aNo
significant
differences
between
flock
AS1
and
AS2
and
flock
W1
and
W2
and
TBE
risk-
and
TBE
non-risk
areas.
in
TBE
risk
areas
and
447
in
TBE
non-risk
areas.
Seroprevalence
in
goats
was
very
low,
only
0.4%
in
TBE
risk
areas
and
0.5%
in
TBE
non-risk
areas
(Table
6).
In
Thuringia,
452
sheep
sera
were
collected
in
2009,
41
in
dis-
tricts
defined
as
TBE
risk
areas
and
411
in
districts
defined
as
TBE
non-risk
areas.
A
total
of
828
goat
sera
was
collected
in
2009,
153
in
TBE
risk
areas
and
675
in
TBE
non-risk
areas.
The
seroprevalence
was
12.8%
in
sheep,
2.0%
in
TBE
risk
areas
and
13.9%
in
TBE
non-
risk
areas
and
1.9%
in
goats,
0.6%
in
TBE
risk
areas
and
2.2%
in
TBE
non-risk
areas
(Tables
7
and
8).
In
North
Rhine-Westphalia,
119
sheep
sera
were
collected
in
2009
and
no
TBEV-specific
antibodies
were
detected.
In
Lower
Sax-
ony,
177
sheep
sera
were
collected
in
2009,
and
two
sera
with
TBEV-specific
antibodies
were
detected.
In
Schleswig-Holstein,
31
sheep
sera
were
collected
in
2009,
and
one
serum
with
TBEV-specific
antibodies
was
detected.
In
Mecklenburg-West
Pomerania
184
sheep
sera
were
collected
in
2008,
and
no
TBEV-
specific
antibodies
were
detected.
In
general,
state-level
seroprevalence
rates
in
sheep
and
goats
were
very
low,
and
the
detected
TBEV
seroprevalence
in
sheep
was
higher
than
in
goats.
Baden-Württemberg
Sheep
sera
were
collected
in
9
districts.
TBEV-specific
positive
sera
were
only
detected
in
the
district
Bodenseekreis
(25
out
of
174
examined
sera
corresponding
to
a
seroprevalence
of
14.4%)
(Table
1).
One
hundred
and
seventy
of
these
174
sheep
were
kept
in
4
flocks
with
20
and
more
animals.
The
seroprevalence
in
these
4
flocks
differed
substantially,
ranging
from
0
to
40%
(Table
1).

C.
Klaus
et
al.
/
Ticks
and
Tick-borne
Diseases
3 (2012) 27–
37 31
Table
5
Seroprevalence
of
TBEV-specific
antibodies
in
sheep
collected
in
Bavaria
in
2008
and
2009.
District
Flock
No.
of
sera
No.
of
positive
sera
Seroprev.a(%)
2009
TBE
risk
areas
Ansbach
(1)
3
1
33
Bad
Kissingen
(2)
BK1 52 7 13
BK2
40
9
23
BK3 57
2
4
Different
90
7
8
Bad
Kissingen
(2),
total
239
25
10.5
Bamberg
(3)
52
1
2
Cham
(4) 38 0 0
Coburg
(5) 16 0 0
Forchheim
(6)
8
0
0
Haßberge
(7)
25
0
0
Main-Spessart
(8)
MS1
40
17
43
MS2
23
2
9
MS3
21
4
19
Different
40
15
38
Main-Spessart
(8),
total
124
38
30.6
Miltenberg
(9) 19 0 0
Neustadt/Aisch
(10)
166
12
7.2
Schweinfurt
(11)
30
1
3
Würzburg
(12)
20
1
5
Total
(1–12)
740
79
10.7
2009
TBE
non-risk
area
Dachau
19
0
0
aSignificant
difference
between:
flock
BK2
and
BK3
(p
=
0.004);
flock
MS1
and
MS3
(p
=
0.005).
A
significant
difference
was
found
between
flocks
BS1
and
BS3
(p
=
0.002)
and
between
flocks
BS3
and
BS4
(p
=
0.007).
A
total
of
2240
goat
sera
was
collected
in
17
districts.
Only
102
TBEV
antibody-positive
sera
were
found
(4.6%).
The
highest
seroprevalence
in
goats
was
detected
in
the
districts
Ortenaukreis
(14.4%)
and
Zollernalbkreis
(21.4%)
and
at
a
much
lower
level
in
the
districts
Breisgau-Hochschwarzwald
(4.5%)
and
Emmendingen
(1.8%).
Significant
differences
were
observed
between
Breisgau-
Hochschwarzwald
and
Emmendingen
(p
=
0.008),
Ortenaukreis
(p
=
0.000)
and
Zollernalbkreis
(p
=
0.001)
as
well
as
between
Emmendingen
and
Ortenaukreis
(p
=
0.000)
and
Zollernalbkreis
(p
=
0.000).
In
these
4
districts,
the
goats
were
kept
in
24
flocks
Table
6
Seroprevalence
of
TBEV-specific
antibodies
in
goats
collected
in
Bavaria
in
2003
and
2009.
District Year No.
of
sera No.
of
positive
sera Seroprev.a(%)
2003/2009
TBE
risk
areas
Aschaffenburg 2003 20
0
0
Aschaffenburg
2009
35
0
0
Bamberg
2003
17
0
0
Bayreuth
2003
6
0
0
Forchheim
2003
8
0
0
Kronach
2003
28
0
0
Main-Spessart
2003
37
0
0
Miltenberg
2003
12
0
0
Neustadt
an
der
Aisch
2003
48
1
2
Neustadt
an
der
Aisch
2009
6
0
0
Regen
2003
11
0
0
Regensburg
2003
17
0
0
Regensburg
2009
4
0
0
Rosenheim
2003
1
0
0
Rottal-Inn
2003
10
0
0
Straubing-Bogen 2003
15
0
0
Total
275
1
0.4
2003/2009
TBE
non-risk
areas
Erding
2003
53
1
2
Fürstenfeldbruck 2003 15 0
0
Landsberg
am
Lech
2003
54
0
0
Miesbach
2003
129
0
0
München
2003
26
0
0
Ostallgäu
2009
3
0
0
Unterallgäu 2003
32
0
0
Weilheim-Schongau 2003
138
1
0.7
Total
450
2
0.4
aNo
significant
difference
between
TBE
risk-
and
TBE
non-risk
areas.

32 C.
Klaus
et
al.
/
Ticks
and
Tick-borne
Diseases
3 (2012) 27–
37
Table
7
Seroprevalence
of
TBEV-specific
antibodies
in
sheep
collected
in
2008
and
2009
in
Thuringia,
North
Rhine-Westphalia,
Lower
Saxony,
Schleswig-Holstein,
and
Mecklenburg-
West
Pomerania.
District
No.
of
sera
No.
of
positive
sera
Seroprev.a(%)
Thuringia,
TBE
risk
areas
Gera 23
0
0
Hildburghausen
7
1
14
Saale-Orla-Kreis 6
0
0
Saalfeld-Rudolstadt
5
0
0
Sonneberg
0
0
0
Total 41 1 2
Thuringia,
TBE
non-risk
areas
(1)
Eichsfeld 19
0
0
Eisenach
116
19
16.4
Erfurt
1
0
0
Greiz
72
9
13
Kyffhäuserkreis 55
0
0
Nordhausen 1
0
0
Schmalkalden-Meiningen
28
10
36
Suhl 9 0 0
Unstrut-Hainich-Kreis
50
0
0
Wartburgkreis
60
19
32
Total
411
57
13.9
North
Rhine-Westphalia,
TBE
non-risk
areas
(2)
Coesfeld
18
0
0
Minden-Lübbecke 20 0
0
Paderborn
50
0
0
Rhein-Erftkreis 9
0
0
Rhein-Sieg-Kreis
21
0
0
Wesel
1
0
0
Total
119
0
0
Lower
Saxony,
TBE
non-risk
areas
(3)
Braunschweig
25
0
0
Celle 30
1
3
Diepholz
7
0
0
Gifhorn
23
0
0
Hannover 92 1
1
Total 177
2
1.1
Schleswig-Holstein,
TBE
non-risk
areas
(4)
Ostholstein
20
1
5
Segeberg
11
0
0
Total 31 1 3
Mecklenburg-West
Pomerania,
TBE
non-risk
area
(5)
Nordvorpommern
184
0
0
Total 184
0
0
Total,
TBE
non-risk
areas
(1–5)
922 60
6.5
aSignificant
differences
between:
TBE
risk-
and
TBE
non-risk
areas
in
Thuringia
(p
=
0.046).
with
18
and
more
animals.
The
seroprevalence
in
these
24
flocks
differed
considerably,
ranging
from
0
to
43%
(Table
2).
In
addition
to
the
serological
results,
1700
ticks
were
collected
and
examined
in
the
districts
Emmendingen
(n
=
526)
and
Orte-
naukreis
(n
=
1174)
with
only
one
female
tick
(district
Ortenaukreis)
found
to
be
TBEV-positive
(Table
3).
Bavaria
In
2003,
sheep
sera
were
collected
in
40
districts.
At
that
time,
20
of
these
districts
were
classified
as
TBE
risk
areas
and
20
as
TBE
non-
risk
areas.
Twenty-six
TBEV-specific
positive
sera
were
detected
in
TBE
risk
areas
and
22
in
TBE
non-risk
areas.
Seroprevalence
was
3.8%
in
the
districts
classified
as
TBE
risk
areas
and
2.8%
in
TBE
non-risk
areas
(Table
4).
Higher
seroprevalences
were
detected
in
districts
with
20
and
more
examined
sera
per
flock
in
Schweinfurt
(15%),
Rosenheim
(10%),
Landshut
(9%),
Amberg-Sulzbach
(7.4%),
and
Wunsiedel
im
Fichtelgebirge
(6.9%).
In
these
5
districts,
372
sheep
were
kept
in
7
flocks
with
20
and
more
animals.
The
seroprevalences
in
these
7
flocks
ranged
from
4%
(flock
AS2)
to
15%
(flock
S1,
Table
4).
Differences
between
the
2
flocks
located
in
the
same
district
in
Amberg-Sulzbach
(AS1
and
AS2)
and
Wunsiedel
(W1
and
W2)
were
not
significant.
In
2008/9,
sheep
sera
were
collected
in
13
districts,
12
of
them
were
classified
as
TBE
risk
areas
and
one
as
TBE
non-risk
area.
A
total
of
79
TBEV-specific
positive
sera
was
detected
in
TBE
risk
areas
(10.7%)
and
none
in
the
TBE
non-risk
area,
where
however
only
19
sera
were
examined
(Table
5).
Higher
seroprevalences
in
districts
with
20
and
more
exam-
ined
sera
per
flock
were
detected
in
the
districts
Main-Spessart

C.
Klaus
et
al.
/
Ticks
and
Tick-borne
Diseases
3 (2012) 27–
37 33
Table
8
Seroprevalence
of
TBEV-specific
antibodies
in
goats
collected
in
Thuringia
in
2009.
District
No.
of
sera
No.
of
positive
sera
Seroprev.a(%)
TBE
risk
areas
Gera 38
0
0
Hildburghausen
31
1
3
Jena
6
0
0
Saale-Holzland-Kreis
26
0
0
Saale-Orla-Kreis
34
0
0
Saalfeld-Rudolstadt 11 0 0
Sonneberg 7 0
0
Total 153
1
0.7
TBE
non-risk
areas
Altenburger
Land
73
5
7
Eichsfeld
24
1
4
Erfurt 14
0
0
Gotha 20
5
25
Ilmkreis
72
0
0
Kyffhäuserkreis 100 0 0
Nordhausen
103
0
0
Schmalkalden-Meiningen
21
0
0
Sömmerda
52
0
0
Suhl
22
1
5
Unstrut-Hainich-Kreis
21
1
5
Wartburgkreis
66
0
0
Weimarer
Land 87 2
2
Total 675
15
2.2
aNo
significant
difference
between
TBE
risk-
and
TBE
non-risk
areas
(p
=
0.330).
(30.6%)
and
Bad
Kissingen
(10.5%).
In
the
district
Main-Spessart,
84
sheep
were
kept
in
3
flocks
with
20
and
more
animals.
The
sero-
prevalences
in
these
3
flocks
ranged
from
9
to
43%
(Table
5).
The
difference
between
flocks
MS1
and
MS3
is
significant
(p
=
0.005).
In
the
district
Bad
Kissingen,
149
sheep
were
kept
in
3
flocks
with
20
and
more
animals.
The
seroprevalences
in
these
3
flocks
ranged
between
4%
and
23%
(Table
5).
The
difference
between
flocks
BK2
and
BK3
is
significant
(p
=
0.004).
In
2003
and
2009,
goat
sera
were
collected
in
21
districts,
13
of
which
were
defined
as
TBE
risk
areas
and
8
as
TBE
non-risk
areas
at
the
time
of
sampling.
TBEV-specific
positive
sera
were
detected
in
2003
only,
one
in
a
TBE
risk
area
and
2
in
TBE
non-risk
areas
(Table
6).
Thuringia
In
2009,
sheep
sera
were
collected
in
15
districts,
5
classified
as
TBE
risk
areas
and
10
as
TBE
non-risk
areas.
A
total
of
58
TBEV-
specific
positive
sera
was
detected,
only
one
in
a
TBE
risk
area
(2%)
and
57
in
TBE
non-risk
areas
(13.9%,
Table
7).
The
differ-
ences
are
significant
(p
=
0.046).
Higher
seroprevalences
in
districts
with
20
and
more
examined
sera
were
detected
in
the
districts
Schmalkalden-Meiningen
(36%),
Wartburgkreis
(32%),
and
Eise-
nach
(16.4%).
In
each
of
these
4
districts,
all
sheep
belonged
to
one
flock.
A
total
of
828
goat
sera
was
collected
in
2009
in
20
districts,
7
districts
were
TBE
risk
areas
and
13
were
TBE
non-risk
areas.
Seroprevalence
was
0.7%
in
TBE
risk
areas
and
2.2%
in
TBE
non-risk
areas
(Table
8).
The
differences
are
not
significant.
Federal
states
with
no
defined
TBE
risk
areas
A
total
of
119
sheep
sera
collected
in
6
districts
of
North
Rhine-Westphalia
in
2009
were
examined
(Table
7).
All
sera
were
found
to
be
TBEV
antibody-negative.
The
same
result
was
found
in
Mecklenburg-West
Pomerania
(one
district,
184
sera).
In
Lower
Saxony
(5
districts,
177
sera),
only
2
sera
were
TBEV-specific
positive
(1.1%),
and
in
Schleswig-Holstein
(2
districts,
31
sera),
only
one
serum
was
TBEV-specific
positive
(3%).
All
geographical
data
(GPS)
of
districts
where
sera
were
col-
lected
are
listed
in
Table
9
.
Discussion
In
general,
TBEV
seroprevalence
was
low
on
the
state
level
and
was
lower
in
goats
than
in
sheep.
Interestingly,
the
highest
TBEV
seroprevalence
was
detected
in
sheep
in
Thuringia
(Table
7).
In
Baden-Wuerttemberg,
positive
sera
were
found
in
sheep
only,
all
of
them
in
the
district
Bodenseekreis.
Four
flocks
with
20
and
more
sheep
were
involved
and
TBEV
seroprevalence
ranged
between
0%
and
40%
and
confirmed
the
patchy
pattern
of
TBEV
foci.
In
goats
in
Baden-Württemberg,
4
districts
were
of
higher
impor-
tance,
Breisgau-Hochschwarzwald,
Emmendingen,
Ortenaukreis,
and
Zollernalbkreis,
and
24
flocks
with
18
and
more
goats
were
examined.
Interestingly,
seroprevalences
ranged
from
0%
to
43%
(Table
2).
The
highest
seroprevalences
were
observed
not
only
in
flocks
in
the
districts
Ortenaukreis
and
Zollernalbkreis,
but
also
in
the
districts
Breisgau-Hochschwarzwald
and
Emmendingen
where
the
seroprevalence
of
goats
in
relation
with
the
whole
district
was
low
(Table
2).
To
obtain
reliable
results,
it
is
therefore
highly
rec-
ommended
to
examine
the
seroprevalence
in
single
flocks
rather
than
the
overall
seroprevalence
in
a
district.
This
may
help
identify
a
very
small,
but
important
TBEV
focus.
Tick
collecting
is
a
well-established
method,
but
is
very
expen-
sive
and
time
consuming.
In
this
study,
only
one
TBEV-positive
tick
was
found
among
the
1174
ticks
collected
in
the
district
Orte-
naukreis
and
none
in
the
district
Emmendingen
(Table
3).
In
both
districts,
however,
presence
of
TBEV
is
very
likely
according
to
test
results
of
the
goat
sera
from
this
area
and
the
number
of
registered
human
TBE
cases
over
the
last
years.
Detection
of
only
one
TBEV-
positive
tick
in
the
district
Ortenaukreis
and
none
in
the
district
Emmendingen
illustrates
the
problem
of
time-consuming
tick
col-
lection
in
the
presence
of
low
virus
prevalence,
even
in
TBEV
risk
areas.
In
Bavaria,
higher
seroprevalences
in
sheep
sera
were
detected
in
only
5
districts,
Schweinfurt
(15%),
Rosenheim
(10%),
Landshut
(9%),
Amberg-Sulzbach
(7.4%),
and
Wunsiedel
im
Fichtelgebirge
(7%)
in
2003.
Seven
flocks
with
20
and
more
animals
were
exam-
ined,
and
the
seroprevalence
ranged
between
4%
(flock
AS2)
and
15%
(flock
S1,
Table
4),
which
is
comparable
to
the
results
obtained
in
Baden-Wuerttemberg,
where
also
remarkable
differences
were
observed
between
flocks
of
the
same
district
(Table
1).
In
2008/9,
high
overall
seroprevalences
were
observed
in
the
districts
Main-
Spessart
(30.6%)
and
Bad
Kissingen
(10.5%).
In
2003
in
contrast,
a
flock
of
39
sheep
was
examined
in
the
district
Bad
Kissingen
revealing
only
one
TBEV
antibody-positive
serum
and
a
low
sero-
prevalence
of
3%.
The
district
Main-Spessart
was
not
examined.
In
2008/9,
3
flocks
were
tested
in
each
of
the
2
districts,
and
the
sero-
prevalences
of
the
individual
flocks
differed
considerably
ranging
from
9%
to
43%
in
the
district
Main-Spessart
and
from
4%
to
23%
in
the
district
Bad
Kissingen
(Table
5).
It
can
be
assumed
that
there
are
small
TBEV
foci
in
these
2
districts.
For
further
investigations
it
is
recommended
to
retest
these
flocks
and
examine
ticks
in
their
immediate
vicinity
because
TBEV
foci
can
be
very
small
and
cross
reactions
with
other
flaviviruses
in
the
SNT
cannot
be
completely
excluded,
especially
if
only
seroconversion
occurs
and
no
clinical
signs
are
seen
in
sheep
(such
as
Louping
ill)
or
in
goats.
In
general,
seroprevalence
in
goats
in
the
examined
districts
in
Bavaria
was
very
low.
In
2003
(677
sera
examined)
and
2009
(48
sera
examined),
only
3
positive
sera
were
found,
one
in
Neustadt
an
der
Aisch,
Erding,
and
Weilheim-Schongau
each.
Therefore,
no
epidemiological
conclusions
were
possible.
This
is
in
contrast
to

34 C.
Klaus
et
al.
/
Ticks
and
Tick-borne
Diseases
3 (2012) 27–
37
Table
9
GPS
data
of
the
districts
where
sera
were
collected.
Country
District
Principal
town
N
E
Baden-Wuerttemberg
Alb-Donau-Kreis
Ulm
48◦2358.64 9◦5947.79
Biberach Biberach 48◦0547.43 9◦4047.63
Bodenseekreis Friedrichshafen
47◦3907.08 9◦2842.55
Breisgau/HS
Freiburg
47◦5949.87 7◦5113.56
Emmendingen Emmendingen
48◦0718.87 7◦5050.47
Esslingen
Esslingen
48◦4427.95 9◦1828.99
Freiburg
Freiburg
47◦5949.87 7◦5113.56
Konstanz
Konstanz
47◦3942.97 9◦1020.73
Lörrach Lörrach 47◦3712.13 7◦3553.85
Ludwigsburg
Ludwigsburg
48◦5406.88 9◦1135.75
Ortenaukreis Offenburg
48◦2804.75 7◦5632.59
Ostalbkreis
Aalen
48◦5009.50 10◦0523.68
Ravensburg
Ravensburg
47◦4703.48 9◦3635.75
Reutlingen
Reutlingen
48◦2927.21 9◦1221.03
Schwäbisch-Hall Schwäbisch-Hall
49◦0639.53 9◦4426.83
Sigmaringen Sigmaringen
48◦0508.72 9◦1259.82
Tübingen
Tübingen
48◦3122.45 9◦0307.55
Tuttlingen Tuttlingen 47◦5859.08 8◦4909.37
Waldshut
Waldshut
47◦3725.27 8◦1251.32
Zollernalbkreis Balingen
48◦1631.75 8◦5118.39
Bavaria
Altötting
Altötting
48◦1235.04 12◦4412.17
Amberg
Amberg
49◦2553.87 11◦5018.08
Amberg-Sulzbach
Amberg
49◦2553.87 11◦5018.08
Ansbach Ansbach 49◦1507.85 10◦3000.82
Aschaffenburg
Aschaffenburg
49◦5822.80 9◦0856.32
Bad
Kissingen Bad
Kissingen
50◦1155.05 10◦0433.22
Bad
Tölz-Wolfratshausen
Bad
Tölz
47◦4542.13 11◦3352.44
Bamberg
Bamberg
49◦5339.18 10◦5307.90
Bayreuth
Bayreuth
49◦5629.61 11◦3415.71
Cham
Cham
49◦1309.12 12◦4003.32
Coburg Coburg 50◦1527.98 10◦5757.71
Dachau
Dachau
48◦1533.82 11◦2603.51
Deggendorf Deggendorf
48◦5001.22 12◦5738.33
Ebersberg
Ebersberg
48◦0438.45 11◦5814.55
Eichstätt
Eichstätt
48◦5327.75 11◦1106.89
Erding Erding 48◦1822.56 11◦5425.30
Erlangen
Erlangen
49◦3559.77 11◦0022.68
Erlangen-Höchstadt Erlangen
49◦3559.77 11◦0022.68
Forchheim
Forchheim
49◦4311.54 11◦0330.24
Freising
Freising
48◦2411.03 11◦4456.93
Freyung-Grafenau
Freyung
48◦4826.37 13◦3249.34
Fürstenfeldbruck
Fürstenfeldbruck
48◦1039.93 11◦1519.31
Garmisch-Partenkirchen Garmisch-
Partenkirchen 47◦2932.43 11◦0510.43
Haßberge
Hassfurt
50◦0153.54 10◦3024.26
Kronach Kronach
50◦1424.23 11◦1924.18
Landsberg
am
Lech
Landsberg
am
Lech
48◦0252.50 10◦5256.91
Landshut
Landshut
48◦3219.76 12◦0906.57
Main-Spessart
Karlstadt
49◦5734.17 9◦4554.10
Miesbach
Miesbach
47◦4523.84 11◦5104.82
Miltenberg
Miltenberg
49◦4211.66 9◦1525.52
Mühldorf
Mühldorf
48◦1420.05 12◦3139.59
München
München
48◦0820.85 11◦3448.67
Neuburg-Schrobenhausen
Neuburg
an
der
Donau
48◦4409.43 11◦1103.58
Neumarkt
Oberpfalz
Neumarkt/Oberpfalz
49◦1656.24 11◦2725.68
Neustadt
an
der
Aisch
Neustadt
an
der
Aisch
49◦3442.14 10◦3619.23
Neustadt
an
der
Waldnaab
Neustadt
a.d.
Waldnaab
49◦4355.47 12◦1030.54
Neu-Ulm
Neu-Ulm
48◦2302.79 10◦0035.06
Oberallgäu
Sonthofen
47◦3057.28 10◦1652.38
Ostallgäu
Marktoberdorf
47◦4638.98 10◦3705.09
Passau Passau
48◦3424.64 13◦2750.10
Regen
Regen
48◦5810.12 13◦0740.21
Regensburg
Regensburg
49◦0059.37 12◦0603.25
Rosenheim
Rosenheim
47◦5121.88 12◦0726.46
Roth
Roth
49◦1044.13 11◦0117.45
Rottal-Inn Pfarrkirchen
48◦2555.06 12◦5617.37
Schwandorf
Schwandorf
49◦1938.81 12◦0631.38
Schweinfurt
Schweinfurt
50◦0256.78 10◦1352.15
Starnberg
Starnberg
48◦0023.07 11◦1748.52
Straubing-Bogen
Straubing
48◦5247.72 12◦3413.00
Unterallgäu
Mindelheim
48◦0212.93 10◦2958.52
Weilheim-Schongau
Weilheim/Obb.
47◦5027.06 11◦0831.84
Würzburg
Würzburg
49◦4739.32 9◦5538.96
Wunsiedel
im
Fichtelgebirge Wunsiedel
im
Fichtelgebirge
50◦0219.61 12◦0018.64

C.
Klaus
et
al.
/
Ticks
and
Tick-borne
Diseases
3 (2012) 27–
37 35
Table
9
(Continued)
Country
District
Principal
town
N
E
Lower
Saxony Ostholstein
Eutin
54◦0811.73 10◦3657.40
Segeberg
Bad
Segeberg
53◦5610.91 10◦1848.27
Mecklenburg-West
Pomerania
Nordvorpommern
Grimmen
54◦0638.14 13◦0220.39
North
Rhine-Westphalia
Coesfeld
Coesfeld
51◦5642.87 7◦1012.01
Minden-Lübbecke
Minden
52◦1723.95 8◦5519.77
Paderborn
Paderborn
51◦4254.91 8◦4507.67
Rhein-Erftkreis
Bergheim
50◦5718.19 6◦3821.97
Rhein-Sieg-Kreis
Siegburg
50◦4755.56 7◦1228.92
Wesel Wesel 51◦3938.98 6◦3632.96
Thuringia
Altenburger
Land
Altenburg
50◦5931.86 12◦2639.58
Eichsfeld
Heiligenstadt
51◦2243.39 10◦0801.14
Eisenach
Eisenach
50◦5827.84 10◦1930.82
Erfurt
Erfurt
50◦5825.45 11◦0120.64
Gera Gera
50◦5249.39 12◦0452.37
Gotha Gotha
50◦5712.30 10◦4352.12
Greiz Greiz
50◦3918.59 12◦1153.62
Hildburghausen
Hildburghausen
50◦2528.05 10◦4356.94
Ilmkreis Arnstadt 50◦5016.49 10◦5638.80
Jena
Jena
50◦5537.20 11◦3513.24
Kyffhäuserkreis
Bad
Frankenhausen
51◦2115.87 11◦0617.51
Nordhausen
Nordhausen
51◦3219.90 10◦4027.52
Saale-Holzland-Kreis
Eisenberg
50◦5805.51 11◦5402.58
Saale-Orla-Kreis
Schleiz
50◦3440.15 11◦4849.45
Saalfeld-Rudolstadt
Rudolstadt
50◦4303.87 11◦1945.65
Schmalkalden-Meiningen Meiningen 50◦3351.24 10◦2454.64
Sömmerda
Sömmerda
51◦0934.00 11◦0707.40
Sonneberg Sonneberg
50◦2124.69 11◦1054.77
Suhl
Suhl
50◦3641.19 10◦4131.35
Unstrut-Hainich-Kreis
Mühlhausen
51◦1226.08 10◦2714.56
Wartburgkreis Bad
Salzungen 50◦4842.79 10◦1356.25
Weimarer
Land
Apolda
51◦0132.32 11◦3059.10
our
earlier
examinations
in
Thuringia
(Klaus
et
al.,
2010c)
and
the
described
results
in
Baden-Wuerttemberg
with
seroprevalences
between
0%
and
43%
(Table
2).
It
is
possible
that
the
examined
goats
in
Bavaria
did
not
live
in
a
TBEV
focus.
In
some
cases,
it
is
also
pos-
sible
that
contact
to
TBEV
causes
only
a
short
immune
response
which
is
no
longer
detectable
after
a
few
weeks.
As
our
knowledge
about
the
longevity
of
TBEV
antibodies
and
possible
differences
between
sheep
and
goats
in
this
respect
is
still
very
limited,
these
differences
cannot
be
interpreted
conclusively.
Interestingly,
high
seroprevalences
were
detected
in
Thuringia
(Table
7).
Some
decades
ago,
TBEV
occurred
in
several
districts
(Süss
et
al.,
1992),
and
it
is
possible
that
some
TBEV
foci
have
become
extinct
(Klaus
et
al.,
2010a)
and
others
have
re-emerged
(Robert
Koch
Institute,
2011).
In
TBE
risk
areas,
only
in
the
district
Hild-
burghausen,
one
of
7
sheep
sera
was
TBEV-positive.
In
contrast,
in
TBE
non-risk
areas,
3
flocks
with
a
remarkably
high
seroprevalence
were
found
in
Eisenach
(16.4%),
Schmalkalden-Meiningen
(36%),
and
Wartburgkreis
(32%)
(Table
7).
These
positive
sheep
sera
may
indicate
so
far
undetected
TBEV
foci
as
shown
in
Suhl
in
previ-
ous
examinations
(Klaus
et
al.,
2010c).
TBEV
antibody-positive
goat
sera
were
detected
in
Gotha
(n
=
5)
and
Altenburger
Land
(n
=
5),
which
are
also
TBE
non-risk
areas;
however,
the
number
of
investi-
gated
sera
in
these
flocks
was
too
small
to
draw
any
epidemiological
conclusions.
These
results
in
TBE
non-risk
areas
in
Thuringia,
a
state
with
a
very
interesting
TBEV
history
(Süss
et
al.,
1992),
should
encour-
age
additional
investigations
in
the
future,
for
example,
to
retest
the
flocks
in
2
or
3
years
and
–
in
case
of
positive
samples
–
to
examine
ticks
in
the
vicinity
of
these
flocks,
as
TBEV
foci
can
be
very
small.
Cross
reactions
with
other
Flaviviridae
in
the
SNT
can-
not
be
excluded
completely
and
may
cause
a
certain
number
of
false-positive
results.
However,
no
potential
cross-reactive
viral
infection
in
sheep
or
goats,
such
as
Louping
ill,
is
known
in
Thuringia
so
far.
To
improve
the
results
from
TBE
non-risk
areas
with
only
some
single
autochthonous
human
TBE
cases,
511
sheep
sera
from
North
Rhine-Westphalia,
Lower
Saxony,
Schleswig-Holstein,
and
Mecklenburg-West
Pomerania
were
examined
and
only
3
TBEV-
specific
positive
sera
were
detected
(0.6%).
In
conclusion,
positive
sera
detected
by
our
test
system
(ELISA
screening
and
SNT
confir-
mation)
may
indicate
unexpected
TBEV
foci
in
regions
currently
defined
as
TBEV
non-risk
areas.
In
Baden-Württemberg
and
Bavaria,
a
considerable
number
of
human
TBE
cases
occurred
in
the
past
10
years
(Robert
Koch
Institute,
2007,
2011),
and
there
are
quite
clear
differences
between
the
districts
where
sera
were
collected
that
are
classified
as
TBE
risk
areas
(84
in
2008
and
88
cases
in
2009
in
the
examined
districts
in
Baden-Württemberg
and
70
in
2003
and
92
cases
in
2009
in
the
examined
districts
in
Bavaria)
and
TBE
non-risk
areas,
where
the
numbers
were
very
low
(4
in
2003
and
5
in
2009
in
the
exam-
ined
districts
in
Bavaria).
The
lowest
number
of
human
TBE
cases
was
registered
in
Thuringia
(2
in
2009
in
districts
classified
as
TBE
risk
areas
and
2
in
districts
classified
as
TBE
non-risk
areas
where
sera
were
collected).
In
many
cases,
our
serological
results
confirm
the
definition
as
TBE
risk
area,
e.g.
in
the
districts
Ortenaukreis,
Bodenseekreis,
Breisgau-Hochschwarzwald,
Emmendingen,
and
Zollernalbkreis
in
Baden-Württemberg
or
Amberg-Sulzbach
and
Main-Spessart
in
Bavaria.
However,
in
some
cases,
our
serological
results
are
in
contrast
to
the
low
number
of
confirmed
TBE
cases,
e.g.
in
Bad
Kissingen,
Rosenheim,
Landshut,
Schweinfurt,
and
Wun-
siedel/Fichtelgebirge
in
Bavaria
or
Hildburghausen
in
Thuringia,
or
to
the
definition
as
TBE
risk
area,
e.g.
in
Altenburger
Land,
Eise-
nach,
Schmalkalden-Meiningen
and
Wartburgkreis
in
Thuringia.
It
is
therefore
recommended
to
retest
sheep
and
goat
sera
of
these
flocks
with
TBEV-specific
titers
in
TBE
non-risk
areas
after
one
or
two
years
in
order
to
identify
changes
of
the
TBEV-specific
serocon-
version
rate
and
to
observe
antibody
titers
over
a
longer
period.

36 C.
Klaus
et
al.
/
Ticks
and
Tick-borne
Diseases
3 (2012) 27–
37
The
presented
data
show
that
the
examination
of
goat
and
sheep
sera
could
be
a
helpful
additional
tool
for
analyzing
the
risk
of
get-
ting
infected
with
TBEV
by
tick
bite
in
a
given
area,
especially
when
high
TBE
vaccination
rates
in
humans
cause
a
decrease
in
the
num-
ber
of
registered
TBE
cases
as
observed
in
Austria.
This
topic
has
also
been
discussed
in
Switzerland,
and
a
high
throughput
proce-
dure
for
tick
surveys
was
proposed
to
solve
the
problem
(Gäumann
et
al.,
2010).
Other
free-ranging
animals
may
also
be
suitable
as
sentinels
for
epidemiological
observations
on
TBEV,
for
example,
wild
rodents
(Achazi
et
al.,
2011),
foxes
(Wurm
et
al.,
2000),
or
roe
deer
as
soon
as
the
problem
of
sample
collection
in
the
field
is
solved.
The
reasons
for
the
patchy
pattern
of
TBEV
occurrence
with
very
small
to
large
areas
are
not
quite
clear
up
to
now.
Never-
theless,
grazing
animals
that
live
in
a
specific
area
might
have
a
closer
contact
to
TBEV
because
of
acquiring
many
tick
bites
and
could
therefore
be
suitable
as
sentinels
and
as
an
early
warn-
ing
system
for
the
presence
of
TBEV.
One
seropositive
goat
was
found
by
Klaus
et
al.
(2010c)
in
the
district
Suhl
(Thuringia),
and
a
TBEV
endemic
area
was
described
in
the
district
Bodenseekreis
by
using
sheep
as
sentinels
(Klaus
et
al.,
2010b).
In
general,
in
all
states
examined
in
the
frame
of
this
study,
seroprevalence
in
sheep
was
higher
than
in
goats.
This
may
be
due
to
the
fact
that
TBEV
immunity
in
goats
after
infection
can
be
very
short,
and
TBEV
immunity
in
sheep
is
detectable
over
a
longer
time
period.
Sera
of
goats
may
therefore
be
helpful
to
detect
a
new
active
TBEV
focus,
and
sheep
sera
may
be
even
more
suitable
to
keep
a
region
under
surveillance
and
to
differentiate
the
regional
patchy
pattern
of
TBEV
foci.
For
antibody
detection,
it
is
of
high
importance
that
all
TBEV
antibody-positive
samples
prescreened
by
ELISA
are
re-tested
by
SNT
as
gold
standard
to
minimize
the
number
of
false-positive
results
because
a
large
number
of
ELISA
results
are
borderline,
and
positive
results
are
non-specific
and
often
cannot
be
confirmed
by
SNT.
However,
this
ELISA
kit
is
very
suitable
for
sera
screening
and
significantly
reduces
the
number
of
sera
that
need
additional
testing
in
the
SNT.
In
conclusion,
seroprevalence
in
free-ranging
animals,
espe-
cially
in
sheep
and
goats,
can
be
a
suitable
and
valuable
additional
tool
to
identify
a
TBEV
focus
in
its
smaller
or
larger
patchy
pat-
tern
and
will
help
to
describe
the
epidemiological
situation.
Further
virological
and
serological
examinations
should
be
carried
out
to
obtain
more
information
on
the
development,
the
spread
and
any
changes
of
TBEV
foci
in
space
and
time
in
a
given
area
and
the
longevity
of
TBEV
antibodies
in
sheep
and
goats.
Acknowledgements
The
authors
are
thankful
to
Katja
Bauer,
Angela
Dram-
burg,
Eva-Maria
Franke,
Elisabeth
Hasse,
Christian
Korthase,
and
Doreen
Reichelt
for
their
excellent
technical
assistance.
We
wish
to
thank
Jens
Böttcher
(Tiergesundheitsdienst
Bay-
ern
e.V.,
Grub/Poing),
Thomas
Miller
(Staatliches
Tierärztliches
Untersuchungsamt,
Aulendorf),
Karl-Heinz
Bogner
(Bayerisches
Landesamt
für
Gesundheit
und
Lebensmittelsicherheit,
Erlan-
gen),
Wilfried
Adams
and
Cordula
Köß
(Landwirtschaftskammer
Nordrhein-Westfalen,
Münster),
and
Martin
Ganter
(University
of
Veterinary
Medicine
Hannover,
Foundation)
for
their
patience
in
collecting
sera.
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