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Veterinary Microbiology 167 (2013) 394–402
Contents lists available at ScienceDirect
Veterinary Microbiology
journal homepage: www.elsevier.com/locate/vetmic
Age-related and regional differences in the prevalence of
hepatitis E virus-specific antibodies in pigs in Germany
Andi Krumbholz a,b,1,*, Sebastian Joel b,1, Anne Neubert b, Paul Dremsek c,
Ralf Dürrwald d, Reimar Johne e, Andreas Hlinak f, Mario Walther g,
Jeannette Lange b, Peter Wutzler b, Andreas Sauerbrei b,
Rainer G. Ulrich c,2, Roland Zell b,2
a
Institute for Infection Medicine, Christian Albrecht University of Kiel and University Medical Center Schleswig-Holstein, Campus Kiel,
24105 Kiel, Germany
b
Department of Virology and Antiviral Therapy, Jena University Hospital, Friedrich Schiller University Jena, 07740 Jena, Germany
c
Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Institute for Novel and Emerging Infectious Diseases,
17493 Greifswald-Insel Riems, Germany
d
IDT Biologika GmbH, 06861 Dessau Rosslau, Germany
e
Federal Institute for Risk Assessment, 10589 Berlin, Germany
f
Landeslabor Berlin-Brandenburg, 15236 Frankfurt (Oder), Germany
g
Institute of Medical Statistics, Computer Sciences and Documentation, Jena University Hospital, Friedrich Schiller University Jena,
07740 Jena, Germany
A R T I C L E I N F O
A B S T R A C T
Article history:
Received 17 June 2013
Received in revised form 27 September 2013
Accepted 4 October 2013
An increasing number of acute autochthonous human hepatitis E virus (HEV)-infections
was noticed in Germany and other developed countries, most likely the result of a zoonotic
virus transmission from pig, wild boar and deer. Currently there is still a lack of profound
data concerning the actual prevalence of HEV-specific antibodies in domestic pig herds in
Germany, in particular for regions with high pig density, and its age-dependency.
2273 domestic pig sera were collected in 2011 mainly from Bavaria, North RhineWestphalia and Lower Saxony from areas having a high pig density. Initially, 420 randomly
selected pig sera were tested in three commercially available and in two in-house HEVantibody ELISAs. 43.6% (183/420) to 65.5% (275/420) of the sera were demonstrated to be
reactive against human pathogenic HEV genotypes 1 and/or 3. The majority of sera reacted
only weakly or not at all with the rat HEV antigen with very few sera showing a stronger
reactivity to this antigen compared to the genotype 3 antigen. The results of all three HEVIgG tests, i.e. the PrioCHECK1 HEV Ab porcine ELISA kit, the ID Screen1 Hepatitis E Indirect
Multi-species ELISA kit and the genotype 3 in-house ELISA were in good accordance.
Therefore, the remaining sera were tested using the PrioCHECK1 HEV Ab porcine ELISA
kit. Samples with a borderline result were finally determined by application of the
conjugate-modified recomLine HEV IgG assay. A total of 1065 of the 2273 sera (46.9%) were
found to be anti-HEV IgG-positive. While 38.4% (306/796) of fatteners (age between 3 and
9 months) exhibited HEV-specific antibodies, 51.4% (759/1477) of sows (age older than 9
months) exhibited anti-HEV antibodies (P < 0.001). Fatteners kept in Southern Germany
had a significantly higher HEV IgG prevalence compared to fatteners kept in the high pig
Keywords:
Germany
Domestic pig
HEV
IgG
Regional variations
Age-dependency
* Corresponding author at: Institute for Infection Medicine, Christian Albrecht University of Kiel and University Medical Center Schleswig-Holstein,
Campus Kiel, Brunswiker Strasse 4, 24105 Kiel, Germany. Tel.: +49 431 597 3306; fax: +49 431 597 3285.
E-mail address: [email protected] (A. Krumbholz).
1
Andi Krumbholz and Sebastian Joel contributed equally to this study.
2
Rainer G. Ulrich and Roland Zell contributed equally to this study.
0378-1135/$ – see front matter ß 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.vetmic.2013.10.001
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395
density federal states North Rhine-Westphalia and Lower Saxony but also in German
federal states with a low pig density.
In conclusion, the present study clearly demonstrates that a high percentage of
domestic pigs in Germany have had contact with HEV. Seroprevalence depends on the
pig’s age and herd origin with the most significant regional variations for fatteners. The
presence of anti-HEV-free herds may indicate that it is feasible to establish and sustain
HEV-free pig herds. HEV seroprevalence still depends on the assay used for testing. This
demonstrates an urgent need for test validation.
ß 2013 Elsevier B.V. All rights reserved.
1. Introduction
At present, Hepatitis E virus (HEV) is the only species
within the genus Hepevirus that comprises the four major
genotypes of the mammalian hepeviruses including the
closely related rabbit HEV, a rat HEV, and the avian HEV
which all represent separate genotypes. The avian HEV
strains appear phylogenetically distinct from the other
hepeviruses and are believed to constitute a further genus
within the family Hepeviridae (Meng et al., 2012). Recently,
novel hepeviruses were identified in Japanese wild boars
(Takahashi et al., 2011) and in ferrets (Raj et al., 2012).
Furthermore, bats (Drexler et al., 2012) and cutthroat trout
(Batts et al., 2011) were identified as carrying hepeviruses.
The human pathogenic HEV genotype 1 is highly
endemic in various developing countries of Asia and Africa
and outbreaks are caused mainly by faecally contaminated
drinking water (Aggarwal, 2011). In addition, this genotype is also responsible for sporadic infections in these
areas (Aggarwal, 2011). HEV genotype 2 has been reported
in human cases in Mexico and Western Africa (Aggarwal,
2011).
For industrialised countries, autochthonous HEV infections – which are documented for humans without travel
history to endemic countries – were demonstrated to be
more common than previously thought. Since 2001, a total
number of 1499 HEV infections have been reported in
Germany (Anonymous, 2013). Even though the proportion
of autochthonous cases has increased from 30 to 78% since
then (Faber et al., 2012), it is not clear whether this is a true
increase or whether the observed increase in symptomatic
HEV infections should be attributed to higher awareness of
physicians. Some support for the latter assumption may
come from recent comparative seroprevalence studies in
Denmark, England and Germany which all found a lower
anti-HEV prevalence than decades before (Christensen
et al., 2008; Ijaz et al., 2009; Sichler et al., 2013).
Autochthonous human HEV infections are associated
with the genotype 3 which is also found in wild boar,
domestic pig, deer, mongoose and rabbit (Adlhoch et al.,
2009a; Brost et al., 2010; Frickmann et al., 2011; Pfefferle
et al., 2012; Preiss et al., 2006). Under experimental
conditions, this genotype has been shown to be able to
cross species barriers and to infect immunologically naı̈ve
pigs and non-human primates (Aggarwal, 2011) but not
rats (Li et al., 2013). HEV genotype 4 is responsible for
sporadic human infections in Asia but is also prevalent in
domestic pigs, wild boars and other mammals in Asia and
was previously found in faecal samples obtained from pigs
in Belgium (Hakze-van der Honing et al., 2011; Meng,
2011). The infection in pigs is asymptomatic and mostly
occurs at an age of 2–4 months (Meng, 2011).
Consumption of raw or undercooked meat products has
been considered as a possible risk factor for zoonotic
transmission of HEV (Meng, 2011). However, a systematic
review revealed an inconsistent association between pork
consumption and HEV seropositivity (Wilhelm et al.,
2011), and in many cases the origin of human HEV
infection remains unknown (Pavio et al., 2010). In addition,
blood transfusion-mediated HEV transmission has been
reported (Vollmer et al., 2012; Wedemeyer et al., 2012).
For Germany – but also for other countries – a higher
prevalence of anti-HEV antibodies has been observed in
persons with direct contact to pigs like veterinarians, pig
farmers and particularly in slaughterers (Krumbholz et al.,
2012; Meng, 2011; Miyamura, 2011). Moreover, it was
previously shown that forestry workers have a higher HEV
seroprevalence rate compared to blood donors (Dremsek
et al., 2012). Thus, in addition to the ingestion of
contaminated meat or to the incorporation of contaminated blood products other transmission routes may be
relevant in case of autochthonous HEV infection.
Several studies in Germany demonstrated the presence
of HEV genotype 3 within the wild boar population as well
as within domestic pigs (Adlhoch et al., 2009b; Baechlein
et al., 2013; Kaci et al., 2008; Schielke et al., 2009; Wenzel
et al., 2011). Furthermore, antibodies against HEV were
detected in about one third of the analysed wild boar sera
(Adlhoch et al., 2009b) and in nearly 50% of the sera
obtained from 1072 domestic pigs (Baechlein et al., 2010).
However, in the latter report application of different
serological assays revealed some discordant results (Baechlein et al., 2010). In a more recent study, sera obtained
from 898 domestic pigs from 10 federal states were
investigated by in-house and commercial IgG antibody
assays resulting in an average seroprevalence of about 43–
45% (Dremsek et al., 2013). However, this study included
only a low number of samples from Lower Saxony and
none from pigs kept in Bavaria and North RhineWestphalia. Together with Lower-Saxony, North RhineWestphalia represents the main focus of the German
fattening industry (see Fig. 1B). About 57% of all domestic
pigs of Germany are kept in both federal states (Anonymous, 2012a).
The major objective of this study was to investigate the
prevalence of HEV-specific antibodies and the presence of
rat HEV-specific antibodies in domestic pigs originating
from high pig density regions of Germany and to identify
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Fig. 1. Geographical origin of the 276 pig herds included in this study (A) and map of the pig density in Germany for 2003 (B). Each herd is indicated in A by
an asterisk. Herds from 11 German federal states were included in this study (B – Bavaria; BB – Brandenburg; BW – Baden- Wuerttemberg; H – Hesse;
LS – Lower Saxony; MW – Mecklenburg-Western Pomerania; NRW – North Rhine-Westphalia; S – Saxony; SA – Saxony-Anhalt; SH – Schleswig-Holstein;
T – Thuringia). The number of analysed pigs/number of pig herds included is indicated in brackets. The figure was generated by the help of stepmap.de. In B
each dot represents 500 pigs. The map was constructed by using the programme DISTRICT based on data purchased from the Wickert Institute, Hildesheim,
Germany (map modified from Krumbholz et al. (2013) and Lange et al. (2013)).
potential age and geographic origin-related differences
therein.
2. Material and methods
2.1. Sera
Between January 2011 and October 2011, a total
number of 4156 domestic pig sera from more than 400
pig breeding and/or pig fattening units from nearly all
German federal states were sent for serological analysis of
influenza virus infection to IDT Biologika GmbH, DessauRosslau, Germany. Sera of piglets (pigs younger than three
months and/or with a body weight below 30 kg) and
follow-up sera were excluded from the final set of sera.
Thus, 2273 sera were included in this study representing
276 German herds with approximately 8.2 pigs per stock
from 11 federal states with the majority of sera coming
from Bavaria, North Rhine-Westphalia and Lower Saxony
(see Fig. 1A). The majority of sera originated from sows
older than nine months (1477, 65%), followed by sera from
fatteners up to nine months of age (796, 35%). Seventyseven out of the 2273 sera (58 from Schleswig-Holstein
and 19 from Hesse) have been already investigated by the
in-house HEV genotype 3 IgG ELISA and two commercial
assays (Dremsek et al., 2013) but are still included in this
setting to achieve a higher degree of comprehensiveness.
For better comparability, four geographical areas were
defined, i.e., Northern Germany (Schleswig-Holstein and
Mecklenburg-Western Pomerania), Northwestern Germany (high pig density federal states North RhineWestphalia and Lower Saxony), Central Germany (Saxony-Anhalt, Hesse, Saxony, Thuringia, and Brandenburg),
and Southern Germany (Bavaria and Baden-Wuerttemberg).
2.2. Selection of a test for seroprevalence study
In the absence of a gold standard for HEV-antibody
testing, a randomly selected set of 418–420 samples from
North Rhine-Westphalia (71%) and Lower Saxony (29%)
was analysed by three commercially available HEV antibody assays (assay A: PrioCHECK1 HEV Ab porcine based
on recombinant ORF2- and 3-derived antigens of genotypes 1 and 3, Prionics AG, Schlieren-Zurich, Switzerland;
assay B: species independent HEV Ab-ELISA kit coated with
recombinant ORF2-derived antigen of genotype 1, Axiom,
Bürstadt, Germany; assay C: ID Screen1 Hepatitis E
Indirect Multi-species ELISA kit, based on recombinant
capsid protein of HEV genotype 3, IDVet Diagnostics,
Montpellier, France) as well as by two in-house IgG ELISAs
based on recombinant carboxy-terminal capsid protein
derivatives of genotype 3 (assay D: (Dremsek et al., 2012,
2013)) and rat HEV (assay E: (Johne et al., 2012)). All
commercial assays were conducted as recommended by
the manufacturers.
If a serum was found to be indeterminate for the
presence of anti-HEV antibodies in one of the assays or if
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397
Fig. 2. HEV antibody prevalence obtained by different assays and determination methods. 418 respectively 420 sera were tested in parallel with assays A, B,
C and D. Borderline results were determined as negative (1), positive (2) or re-evaluated by application of the recomLine assay (3). Seroprevalences are given
for fatteners and sows and for the total group. P-values are based on Fisher’s exact test for comparing the prevalence between fatteners and sows.
results differed between the assays (i.e., deviant result),
this sample was re-tested in a modified commercially
available strip immunoassay which is also based on HEV
genotype 1 and 3 antigens (recomLine HEV IgG assay,
Mikrogen GmbH, Neuried, Germany) by application of
1:7500 diluted peroxidase-conjugated rabbit anti-swine
IgG (H&L) (Rockland Immunochemicals, Gilbertsville, PA,
USA) instead of peroxidase-conjugated anti-human IgG
(Werres, 2010). The recomLine HEV IgG strips were
automatically analysed and evaluated by recomScan II
software (Mikrogen GmbH).
2.3. Seroprevalence study
Assay A was used for testing the residual 1853 serum
samples and to determine the total HEV seroprevalence.
Again, the conjugate-modified recomLine HEV IgG assay
was applied if a serum was found to be indeterminate by
assay A.
2.4. Statistics
The results are presented as numbers and percentages where necessary. Group-specific prevalences of
HEV-specific antibodies together with 95% confidence
interval (CI) were calculated considering the geographical
origin and age of the pig for each sample. For comparison of
the seroprevalence between different groups Fisher’s exact
test was applied. In order to compare the test results
achieved by the different assays, Cohen’s kappa was
calculated as a measure of test agreement (Landis and
Koch, 1977). A kappa between 0.41 and 0.60 indicates a
moderate, between 0.61 and 0.80 a substantial and
between 0.81 and 1.00 an almost perfect agreement
(Landis and Koch, 1977). SPSS 20.0.0 (IBM1) was used for
statistical evaluation of the results. A P-value of 0.05 was
considered as statistically significant.
3. Results
3.1. Comparative determination of HEV-specific antibodies
by different assays
Initially, 420 randomly selected sera were tested in
parallel with the three assays A, B, D and 418 sera with
assay C. For assays A and B different determination
methods were used for borderline results. The overall
prevalence varied between 43.6% and 65.5% depending on
the assay used (see Fig. 2). The calculation of Cohen’s kappa
showed substantial to almost perfect agreement for the
three assays A, C and D (0.757–0.824). The Cohen’s kappa
varied between 0.579 and 0.770 when comparing assay B
to the other three assays (see Table 1). All four assays
demonstrated a significantly higher seroprevalence in
sows compared to fatteners (see Fig. 2).
In the absence of a widely accepted reference test for
HEV serology a modified recomLine HEV IgG assay was
applied to confirm deviant results (i.e., positive/negative;
negative/positive) initially obtained by the four assays. In
this setting, the recomLine HEV IgG test is the only assay
which is not based on the ELISA-technology and allows
measurement of antibodies against discrete antigens of
HEV. This product has been successfully used to measure
HEV seroprevalence in human patient sera with known
infection status (Osterman et al., 2012) and served also as
reference test for the determination of anti-HEV antibodies in pig sera and pig meat juice (Wacheck et al.,
2012b; Werres, 2010). In this study, borderline results
obtained by assays A or B were determined as anti-HEVpositive in order to facilitate test comparison since this
determination method achieved the highest kappa value
(0.824; A2/C). Between 5.1% and 94.9% of deviant test
results were confirmed by the recomLine HEV IgG assay.
Only a minority of deviant results obtained by assay B
were verifiable by the latter test (see supplementary
Table 1).
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Table 1
Presence of HEV-specific antibodies in a randomly selected set of 418–420 sera determined by different antibody assays.
A1 borderline results in assay A were determined as negative.
A2 borderline results in assay A were determined as positive.
A3 borderline results in assay A were re-evaluated by application of strip immunoassay.
B1 borderline results in assay B were determined as negative.
B2 borderline results in assay B were determined as positive.
neg = negative.
pos = positive.
* 418 sera instead of 420 sera were available for testing.
Kappa values indicating test agreement are given in grey boxes.
Parallel testing of all 420 sera using the genotype 3
(assay D) and rat HEV in-house (assay E) ELISAs demonstrated that the majority of pig sera reacted exclusively
with the genotype 3 antigen and few sera demonstrated
almost exclusive reactivity or strong cross-reactivity to the
rat HEV antigen (see supplementary Fig. 1).
3.2. Seroprevalence study
To estimate the seroprevalence of the entire serum
panel, the residual 1853 pig sera were investigated by
assay A. This assay was selected since it showed an
excellent agreement to the independently developed ELISA
C and was previously demonstrated to measure anti-HEVIgG antibodies in pig meat juice and sera reliably (Dremsek
et al., 2013; Wacheck et al., 2012a, 2012b; Werres, 2010).
864 (46.6%) of the residual sera had HEV-specific IgG
antibodies. This refers to 278 out of 681 HEV IgG-positive
fatteners (40.8%) and 586 out of 1172 sows (50.0%).
Borderline results were obtained in 5.6% (103/1853) of the
cases; among them were 2.5% (17/681) fatteners and 7.3%
(86/1172) sows (for details see supplementary Table 2a).
Re-testing of these 103 sera in the modified recomLine IgG
assay resulted in 14 (13.6%) sera which were determined as
positive and 89 (86.4%) sera that revealed a negative result
(supplementary Table 2b).
Together with the above described analysis of the 420
sera, the total of 2273 sera showed an estimated
seroprevalence of 46.9% (supplementary Table 3) with a
range of 38.4% for fatteners and 51.4% for sows. The
difference in HEV seroprevalence between fatteners and
sows was statistically significant (P < 0.001).
The HEV IgG prevalence varied significantly between
41.1% in Northwestern Germany and 51.4% in Southern
Germany (Table 2; P < 0.001). 20.6% of the fatteners in
Northwestern Germany carried HEV-specific IgG antibodies compared to 54.9% of the fatteners in Southern
Germany (P < 0.001). The lowest HEV IgG prevalence was
found in fatteners kept in German federal states
characterised by a low pig density. In contrast, HEV
IgG prevalence of sows was similar between the four
regions irrespective of pig density (P = 0.193; for details
see also Table 2). In 19.9% of the herds no anti-HEV IgG
antibodies were detectable while in 23.2% of the herds
more than 75% of tested pigs carried HEV-specific IgG
antibodies (Fig. 3).
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399
Table 2
Geographical differences in HEV seroprevalence in domestic pigs (2273 sera) from Germany.
Combination of assay A and recomLine IgG assay
95% jCI
Positive/total number tested (%)
Fatteners
(age between 3
and 9 months)
Region
Northern Germany
Northwestern Germany
Central Germany
Southern Germany
2/25 (8.0)
69/335 (20.6)
1/10 (10.0)
234/426 (54.9)
306/796 (38.4)
1.0–26.0
16.4–25.3
0.3–44.5
50.1–59.7
35.0–41.9
Northern Germany
Northwestern Germany
Central Germany
Southern Germany
42/68 (61.8)
307/580 (52.9)
33/67 (49.3)
377/762 (49.5)
759/1477 (51.4)
49.2–73.3
48.8–57.1
36.8–61.8
45.9–53.1
48.8–54.0
Northern Germany
Northwestern Germany
Central Germany
Southern Germany
44/93 (47.3)
376/915 (41.1)
34/77 (44.2)
611/1188 (51.4)
1065/2273 (46.9)
36.9–57.9
37.9–44.4
32.8–55.9
48.5–54.3
44.8–48.9
Total (P < 0.001)*
Sows (>9 months of age)
Region
Total (P = 0.193)*
All pigs
Region
Total (P < 0.001)*
* P-values for comparison of HEV IgG prevalence between the four regions; Northern Germany (Schleswig-Holstein and Mecklenburg-Western
Pomerania), Northwestern Germany (North Rhine-Westphalia and Lower Saxony), Central Germany (Saxony-Anhalt, Hesse, Saxony, Thuringia, and
Brandenburg), Southern Germany (Bavaria and Baden-Wuerttemberg) within the two age groups of pigs and for the total number of pigs, respectively.
Fig. 3. Variation of HEV IgG seroprevalence in different pig herds determined by assay A3 (combination of assay A and modified recomLine IgG assay).
4. Discussion
In the present study, three commercially available and
one in-house assay were compared in order to find an assay
suitable for large scale seroprevalence studies. The best
agreement was found between assays A, C and D and in
particular between assays A and C. The latter tests are based
on recombinantly expressed antigens of HEV genotypes 1
and 3, whereas assay D uses a genotype 3 antigen alone. The
genotype 1-based double-antigen sandwich ELISA (assay B)
was found to give the highest degree of seroreactivity, but a
markedly lower degree of agreement with the other three
assays. Similar results have been reported previously in HEV
seroprevalence studies using pig meat juice and sera
(Dremsek et al., 2013; Wacheck et al., 2012b). Most of the
deviant sera in the current study, that were found to be
positive in assay B but negative in the other assays, were not
verifiable in a (modified) line assay which has been
previously successfully used to measure HEV seroprevalence in human patient sera, pig sera and pig meat juice
(Osterman et al., 2012; Wacheck et al., 2012b; Werres,
2010). It can be argued that sera with isolated anti-HEV IgM
or IgA may be missed in the IgG-specific assays A, C and D.
However, occurrence of isolated anti-HEV IgM in fatteners is
rare (Wacheck et al., 2012b; Werres, 2010). The contribution
of IgA to increased seroreactivity in assay B is not known
and needs further investigation. Previously, assay A was
demonstrated to have good sensitivity and specificity
compared to the time-consuming and expensive line assay
(Wacheck et al., 2012b; Werres, 2010). Thus, this ELISA as
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well as the assays C and D – which exhibited excellent
correlation in results – may be suitable for large scale
seroprevalence studies. Borderline results obtained with
assay A may be clarified by the application of the modified
recomLine assay.
The overall HEV seroprevalence of 46.9% (95% CI: 44.8–
48.9%) observed in this study for more than 2200 domestic
pigs originating from most German federal states is in line
with previous findings concerning a considerably lower
number of domestic pigs from several federal states of
Germany (Baechlein et al., 2010; Dremsek et al., 2013).
Moreover, in the present study an age-dependency of the
seroprevalence was found. Thus, a significantly different
prevalence of HEV-specific antibodies was evident between
fatteners of an age between three and nine months (38.4%)
and sows older than nine months (51.4%). Interestingly, the
observed difference is independent of the assay that was
used as it was found for all four assays in the initial pilot
investigation. Comparable results were reported in a
previous German study based on an in-house assay using
a considerably lower number of pig sera. Therein, 31.3% (30/
96) of fatteners and 50.0% (23/46) of sows presented HEVspecific antibodies (Baechlein et al., 2010). The trend of
higher HEV seroprevalence in sows was further observed in
other European countries (Jimenez de Oya et al., 2011;
Martinelli et al., 2011; Seminati et al., 2008). In contrast,
sows were demonstrated to have the lowest HEV RNA
prevalence compared to younger pigs like fatteners, growers
and weaners (Berto et al., 2012a). The higher HEVseropositivity and the low prevalence of HEV in older pigs
might be explained by the increased probability of exposure
during the lifespan, a phenomenon also discussed for the
situation concerning humans (Faber et al., 2012). Interestingly, in the current study fatteners from Southern Germany
(including Bavaria with spatially high pig density) had a
considerably higher HEV IgG prevalence compared to
fatteners from high pig density federal states North RhineWestphalia and Lower Saxony but also from other German
federal states with low pig density while seroprevalence in
sows from the different regions was similar. The occurrence
of regional differences in HEV seroprevalence has been
described previously (Baechlein et al., 2010; Dremsek et al.,
2013) and may be explained by the different origins of
piglets between both regions. In Northern Germany, a
significant proportion of piglets is imported from Denmark
and The Netherlands while in Southern Germany the
proportion of piglets originating from local breeders is
higher (Anonymous, 2012b). Thus, the detailed sources of
pigs and breeding conditions should be further analysed.
Other authors found considerably higher HEV seroprevalences of 68% and 71% for pigs kept in farms (Baechlein et al.,
2010) or slaughtered in Bavaria (Wacheck et al., 2012b). This
might come from the lower number of samples included in
the previous studies or from different sampling strategies, as
the current study includes sera originally collected for an
influenza survey (Lange et al., 2013).
Interestingly, in nearly 20% of the analysed pig herds no
HEV antibody was detectable (see Fig. 3). Among them
were 30 herds from Bavaria, 16 herds from North RhineWestphalia, seven herds from Lower Saxony and one herd
each from Schleswig-Holstein and Baden-Wuerttemberg.
This provides evidence that HEV-free pig breeding is
generally possible. First insights into the HEV infection
dynamics within farrow-to-finish pig farms were published recently (Casas et al., 2011).
The observed low or almost absent prevalence of rat
HEV-specific antibodies in pigs is in line with previous data
(Dremsek et al., 2013). These findings might indicate the
limitations of the species crossing as reported for rat HEV
(Purcell et al., 2011) and other genotypes (Li et al., 2013).
However, the previous finding of rat HEV-reactive antibodies in forestry workers (Dremsek et al., 2012) also
underlines the necessity for further evaluation of rat HEV
transmission from rats to other mammals.
Taken together, the present study clearly demonstrates
that HEV-specific antibodies show a broad geographical
distribution and age-dependent prevalence within the
domestic pigs of Germany. To our knowledge, this is the
most comprehensive and recent survey done in Germany
comprising samples from the high pig density areas of
Lower Saxony, North Rhine-Westphalia and parts of
Bavaria tested in various HEV antibody assays. This setting
as well as the considerably higher number of analysed pigs
compared to former studies provide evidence of regional
variations of HEV seroprevalence in fatteners in Germany.
A high prevalence of HEV and HEV-specific antibodies in
pigs was also observed in other European countries (Berto
et al., 2012a, 2012b; Breum et al., 2010; Di Bartolo et al.,
2011; Jimenez de Oya et al., 2011; Kaba et al., 2009;
Meader et al., 2010; Rose et al., 2011; Seminati et al., 2008;
Wacheck et al., 2012a). Thus, domestic pigs might
represent one of the most important sources of autochthonous HEV infection in Germany and also in other
European countries. The main focus of future research
should be to follow HEV contaminations along the whole
pig and meat production line and to identify potential pighuman transmission routes.
Conflict of interest
Mikrogen GmbH, Neuried, Germany has delivered assay
A and the HEV recomLine assay free of charge. The ID
Screen1 Hepatitis E Indirect Multi-species ELISA kit (assay
C) was a kind gift of IDVet Diagnostics, Montpellier, France.
Samples were tested independently by Sebastian Joel
(assays A, B and HEV recomLine), Anne Neubert (assay C)
and by Paul Dremsek and Andreas Hlinak (in-house assays
D and E). Mikrogen and IDVet Diagnostics had no influence
on testing of sera and interpretation of test results nor on
the manuscript draft.
Data were presented in parts at the National Symposium on Zoonoses Research, 2012, Berlin, Germany, and at
the 23rd Meeting of the Society for Virology, 2013, Kiel,
Germany.
Acknowledgments
The excellent support by Dr. Silvia Dorn und Michael
Dawideit, Mikrogen, Neuried, Germany, by Kristine
Klewer, IDVet Diagnostics, Montpellier, France, and the
excellent technical assistance of Dörte Kaufmann, Friedrich-Loeffler-Institut, Greifswald-Insel Riems, Germany,
Author's personal copy
A. Krumbholz et al. / Veterinary Microbiology 167 (2013) 394–402
and Dagmar Hübner, Landeslabor Berlin-Brandenburg,
Germany, is cordially acknowledged. Furthermore, the
authors would like to thank Dr. Ulrich Mohn, Roche,
Penzberg, Germany and Dr. Silke Wacheck, LudwigMaximilians-Universität, München, Germany for helpful
advice. In addition, proof-reading and editing of the
manuscript done by Clare Weber, Dessau, Germany, is
kindly acknowledged.
Appendix A. Supplementary data
Supplementary material related to this article can be
found, in the online version, at http://dx.doi.org/10.1016/
j.burns.2013.10.009.
References
Adlhoch, C., Kaiser, M., Pauli, G., Koch, J., Meisel, H., 2009a. Indigenous
hepatitis E virus infection of a plasma donor in Germany. Vox Sanguinis 97, 303–308.
Adlhoch, C., Wolf, A., Meisel, H., Kaiser, M., Ellerbrok, H., Pauli, G., 2009b.
High HEV presence in four different wild boar populations in East and
West Germany. Veterinary Microbiology 139, 270–278.
Aggarwal, R., 2011. Hepatitis E: historical, contemporary and future perspectives. Journal of Gastroenterology and Hepatology 26 (Suppl. 1)
72–82.
Anonymous, 2012a. Landwirtschaft – Viehwirtschaft; November 3, 2012.,
http://www.statistik-portal.de/Statistik-Portal/de_jb11_jahrtab22.asp (Statistische Ämter des Bundes und der Länder).
Anonymous, 2012b. Smågrise overskud og underskud i Tyskland 2006–
2012*.,
http://www.danskesvineproducenter.dk/upload/fckeditor/
Kort%20Tyskland%20Sep12.pdf (Danske Svineproducenter).
Anonymous, 2013. Übermittelte Hepatitis E-Fälle nach Meldekategorie,
Deutschland, Fälle entsprechend der Referenzdefinition des RKI;
Datenstand: 29. 05. 2013; SurvStat., http://www3.rki.de/SurvStat
(Robert Koch-Institut).
Baechlein, C., Schielke, A., Johne, R., Ulrich, R.G., Baumgaertner, W.,
Grummer, B., 2010. Prevalence of Hepatitis E virus-specific antibodies
in sera of German domestic pigs estimated by using different assays.
Veterinary Microbiology 144, 187–191.
Baechlein, C., Seehusen, F., Nathues, H., Beilage, E., Baumgartner, W.,
Grummer, B., 2013. Molecular detection of hepatitis E virus in German
domestic pigs. Berliner und Münchener tierärztliche Wochenschrift
126, 25–31.
Batts, W., Yun, S., Hedrick, R., Winton, J., 2011. A novel member of the
family Hepeviridae from cutthroat trout (Oncorhynchus clarkii). Virus
Research 158, 116–123.
Berto, A., Backer, J.A., Mesquita, J.R., Nascimento, M.S., Banks, M., Martelli,
F., Ostanello, F., Angeloni, G., Di Bartolo, I., Ruggeri, F.M., Vasickova, P.,
Diez-Valcarce, M., Hernandez, M., Rodriguez-Lazaro, D., van der Poel,
W.H., 2012a. Prevalence and transmission of hepatitis E virus in
domestic swine populations in different European countries. BMC
Research Notes 5, 190.
Berto, A., Mesquita, J.R., Hakze-van der Honing, R., Nascimento, M.S., van
der Poel, W.H., 2012b. Detection and characterization of hepatitis E
virus in domestic pigs of different ages in Portugal. Zoonoses and
Public Health 59, 477–481.
Breum, S.O., Hjulsager, C.K., de Deus, N., Segales, J., Larsen, L.E., 2010.
Hepatitis E virus is highly prevalent in the Danish pig population.
Veterinary Microbiology 146, 144–149.
Brost, S., Wenzel, J.J., Ganten, T.M., Filser, M., Flechtenmacher, C., Boehm,
S., Astani, A., Jilg, W., Zeier, M., Schnitzler, P., 2010. Sporadic cases of
acute autochthonous hepatitis E virus infection in Southwest Germany. Journal of Clinical Virology 47, 89–92.
Casas, M., Cortes, R., Pina, S., Peralta, B., Allepuz, A., Cortey, M., Casal, J.,
Martin, M., 2011. Longitudinal study of hepatitis E virus infection in
Spanish farrow-to-finish swine herds. Veterinary Microbiology 148,
27–34.
Christensen, P.B., Engle, R.E., Hjort, C., Homburg, K.M., Vach, W., Georgsen,
J., Purcell, R.H., 2008. Time trend of the prevalence of hepatitis E
antibodies among farmers and blood donors: a potential zoonosis in
Denmark. Clinical Infectious Diseases 47, 1026–1031.
Di Bartolo, I., Ponterio, E., Castellini, L., Ostanello, F., Ruggeri, F.M., 2011.
Viral and antibody HEV prevalence in swine at slaughterhouse in
Italy. Veterinary Microbiology 149, 330–338.
401
Dremsek, P., Joel, S., Baechlein, C., Pavio, N., Schielke, A., Ziller, M.,
Durrwald, R., Renner, C., Groschup, M.H., Johne, R., Krumbholz, A.,
Ulrich, R.G., 2013. Hepatitis E virus seroprevalence of domestic pigs in
Germany determined by a novel in-house and two reference ELISAs.
Journal of Virological Methods 190, 11–16.
Dremsek, P., Wenzel, J.J., Johne, R., Ziller, M., Hofmann, J., Groschup, M.H.,
Werdermann, S., Mohn, U., Dorn, S., Motz, M., Mertens, M., Jilg, W.,
Ulrich, R.G., 2012. Seroprevalence study in forestry workers from
eastern Germany using novel genotype 3- and rat hepatitis E virusspecific immunoglobulin G ELISAs. Medical Microbiology and Immunology 201, 189–200.
Drexler, J.F., Seelen, A., Corman, V.M., Fumie Tateno, A., Cottontail, V.,
Melim Zerbinati, R., Gloza-Rausch, F., Klose, S.M., Adu-Sarkodie, Y.,
Oppong, S.K., Kalko, E.K., Osterman, A., Rasche, A., Adam, A., Muller,
M.A., Ulrich, R.G., Leroy, E.M., Lukashev, A.N., Drosten, C., 2012. Bats
worldwide carry hepatitis E virus-related viruses that form a putative
novel genus within the family Hepeviridae. Journal of Virology 86,
9134–9147.
Faber, M.S., Wenzel, J.J., Jilg, W., Thamm, M., Hohle, M., Stark, K., 2012.
Hepatitis E virus seroprevalence among adults, Germany. Emerging
Infectious Diseases 18, 1654–1657.
Frickmann, H., Gabriel, M., Polywka, S., Bonow, I., Gunther, S., SchmidtChanasit, J., 2011. An unusual cause of elevated liver function tests in
an elderly female. Journal of Clinical Virology 51, 93–95.
Hakze-van der Honing, R.W., van Coillie, E., Antonis, A.F., van der Poel,
W.H., 2011. First isolation of hepatitis E virus genotype 4 in Europe
through swine surveillance in the Netherlands and Belgium. PLoS
ONE 6, e22673.
Ijaz, S., Vyse, A.J., Morgan, D., Pebody, R.G., Tedder, R.S., Brown, D., 2009.
Indigenous hepatitis E virus infection in England: more common than
it seems. Journal of Clinical Virology 44, 272–276.
Jimenez de Oya, N., de Blas, I., Blazquez, A.B., Martin-Acebes, M.A.,
Halaihel, N., Girones, O., Saiz, J.C., Escribano-Romero, E., 2011. Widespread distribution of hepatitis E virus in Spanish pig herds. BMC
Research Notes 4, 412.
Johne, R., Dremsek, P., Kindler, E., Schielke, A., Plenge-Bonig, A., Gregersen,
H., Wessels, U., Schmidt, K., Rietschel, W., Groschup, M.H., Guenther, S.,
Heckel, G., Ulrich, R.G., 2012. Rat hepatitis E virus: geographical
clustering within Germany and serological detection in wild Norway
rats (Rattus norvegicus). Infection, Genetics And Evolution 12, 947–956.
Kaba, M., Davoust, B., Marie, J.L., Barthet, M., Henry, M., Tamalet, C., Raoult,
D., Colson, P., 2009. Frequent transmission of hepatitis E virus among
piglets in farms in Southern France. Journal of Medical Virology 81,
1750–1759.
Kaci, S., Nockler, K., Johne, R., 2008. Detection of hepatitis E virus in
archived German wild boar serum samples. Veterinary Microbiology
128, 380–385.
Krumbholz, A., Lange, J., Dürrwald, R., Walther, M., Müller, T.H., Kühnel, D.,
Wutzler, P., Sauerbrei, A., Zell, R., 2013. Prevalence of antibodies to
European porcine influenza viruses in humans living in high pig
density areas of Germany. Medical Microbiology and Immunology,
http://dx.doi.org/10.1007/s00430-013-0309-y (in press).
Krumbholz, A., Mohn, U., Lange, J., Motz, M., Wenzel, J.J., Jilg, W.,
Walther, M., Straube, E., Wutzler, P., Zell, R., 2012. Prevalence of
hepatitis E virus-specific antibodies in humans with occupational
exposure to pigs. Medical Microbiology and Immunology 201, 239–
244.
Landis, J.R., Koch, G.G., 1977. The measurement of observer agreement for
categorical data. Biometrics 33, 159–174.
Lange, J., Groth, M., Schlegel, M., Krumbholz, A., Wieczorek, K., Ulrich, R.,
Köppen, S., Schulz, K., Appl, D., Selbitz, H.J., Sauerbrei, A., Platzer, M.,
Zell, R., Dürrwald, R., 2013. Reassortants of the Pandemic (H1N1)
2009 Virus and establishment of a novel porcine H1N2 influenza virus
lineage in Germany. Veterinary Microbiology, http://dx.doi.org/
10.1016/j.vetmic.2013.09.024 (in press).
Li, T.C., Yoshizaki, S., Ami, Y., Suzaki, Y., Yasuda, S.P., Yoshimatsu, K.,
Arikawa, J., Takeda, N., Wakita, T., 2013. Susceptibility of laboratory
rats against genotypes 1, 3, 4, and rat hepatitis E viruses. Veterinary
Microbiology 163, 54–61.
Martinelli, N., Luppi, A., Cordioli, P., Lombardi, G., Lavazza, A., 2011.
Prevalence of hepatitis E virus antibodies in pigs in Northern Italy.
Infection Ecology & Epidemiology 1.
Meader, E., Thomas, D., Salmon, R., Sillis, M., 2010. Seroprevalence of
hepatitis E virus in the UK farming population. Zoonoses and Public
Health 57, 504–509.
Meng, X.J., 2011. From barnyard to food table: the omnipresence of
hepatitis E virus and risk for zoonotic infection and food safety. Virus
Research 161, 23–30.
Meng, X.J., Anderson, D.A., Arankalle, V.A., Emerson, S.U., Harrison, T.J.,
Jameel, S., Okamoto, H., 2012. Hepeviridae. In: King, A.M.Q., Adams,
Author's personal copy
402
A. Krumbholz et al. / Veterinary Microbiology 167 (2013) 394–402
M.J., Carstens, E.B., Lefkowitz, E.J. (Eds.), Virus Taxonomy. Academic
Press Elsevier, Amsterdam, pp. 1021–1028.
Miyamura, T., 2011. Hepatitis E virus infection in developed countries.
Virus Research 161, 40–46.
Osterman, A., Vizoso Pinto, M.G., Haase, R., Nitschko, H., Jager, S., Sander,
M., Motz, M., Mohn, U., Baiker, A., 2012. Systematic screening for
novel, serologically reactive Hepatitis E Virus epitopes. Virology
Journal 9, 28.
Pavio, N., Meng, X.J., Renou, C., 2010. Zoonotic hepatitis E: animal reservoirs and emerging risks. Veterinary Research 41, 46.
Pfefferle, S., Frickmann, H., Gabriel, M., Schmitz, N., Gunther, S., SchmidtChanasit, J., 2012. Fatal course of an autochthonous hepatitis E virus
infection in a patient with leukemia in Germany. Infection 40, 451–454.
Preiss, J.C., Plentz, A., Engelmann, E., Schneider, T., Jilg, W., Zeitz, M.,
Duchmann, R., 2006. Autochthonous hepatitis E virus infection in
Germany with sequence similarities to other European isolates. Infection 34, 173–175.
Purcell, R.H., Engle, R.E., Rood, M.P., Kabrane-Lazizi, Y., Nguyen, H.T.,
Govindarajan, S., St Claire, M., Emerson, S.U., 2011. Hepatitis E virus
in rats, Los Angeles, California, USA. Emerging Infectious Diseases 17,
2216–2222.
Raj, V.S., Smits, S.L., Pas, S.D., Provacia, L.B., Moorman-Roest, H., Osterhaus,
A.D., Haagmans, B.L., 2012. Novel hepatitis E virus in ferrets, the
Netherlands. Emerging Infectious Diseases 18, 1369–1370.
Rose, N., Lunazzi, A., Dorenlor, V., Merbah, T., Eono, F., Eloit, M., Madec, F.,
Pavio, N., 2011. High prevalence of Hepatitis E virus in French
domestic pigs. Comparative Immunology, Microbiology and Infectious Diseases 34, 419–427.
Schielke, A., Sachs, K., Lierz, M., Appel, B., Jansen, A., Johne, R., 2009.
Detection of hepatitis E virus in wild boars of rural and urban regions
in Germany and whole genome characterization of an endemic strain.
Virology Journal 6, 58.
Seminati, C., Mateu, E., Peralta, B., de Deus, N., Martin, M., 2008. Distribution of hepatitis E virus infection and its prevalence in pigs on
commercial farms in Spain. Veterinary Journal 175, 130–132.
Sichler, M., Behrens, G., Leitzmann, M., Jilg, W., Wenzel, J.J., 2013. Hepatitis E in Germany – an emerging infection? In: 23rd Annual Meeting
of the Society for Virology, Kiel, Germany, p. 553.
Takahashi, M., Nishizawa, T., Sato, H., Sato, Y., Jirintai, Nagashima, S.,
Okamoto, H., 2011. Analysis of the full-length genome of a hepatitis E
virus isolate obtained from a wild boar in Japan that is classifiable into
a novel genotype. The Journal of General Virology 92, 902–908.
Vollmer, T., Diekmann, J., Johne, R., Eberhardt, M., Knabbe, C., Dreier, J.,
2012. Novel approach for detection of hepatitis E virus infection in
German blood donors. Journal of Clinical Microbiology 50, 2708–
2713.
Wacheck, S., Sarno, E., Martlbauer, E., Zweifel, C., Stephan, R., 2012a.
Seroprevalence of anti-hepatitis E virus and anti-Salmonella antibodies in pigs at slaughter in Switzerland. Journal of Food Protection 75,
1483–1485.
Wacheck, S., Werres, C., Mohn, U., Dorn, S., Soutschek, E., FredrikssonAhomaa, M., Martlbauer, E., 2012b. Detection of IgM and IgG against
hepatitis E virus in serum and meat juice samples from pigs at
slaughter in Bavaria, Germany. Foodborne Pathogens and Disease
9, 655–660.
Wedemeyer, H., Pischke, S., Manns, M.P., 2012. Pathogenesis and treatment of hepatitis E virus infection. Gastroenterology 142 , 1388–1397
e1381.
Wenzel, J.J., Preiss, J., Schemmerer, M., Huber, B., Plentz, A., Jilg, W., 2011.
Detection of hepatitis E virus (HEV) from porcine livers in Southeastern Germany and high sequence homology to human HEV isolates. Journal of Clinical Virology 52, 50–54.
Werres, C., 2010. Entwicklung eines ELISA zum Nachweis von Hepatitis E
Antikörpern aus Serum und Fleischsaft des Schweins. Ludwig-Maximilians-Universität München, Munich.
Wilhelm, B.J., Rajic, A., Greig, J., Waddell, L., Trottier, G., Houde, A., Harris,
J., Borden, L.N., Price, C., 2011. A systematic review/meta-analysis of
primary research investigating swine, pork or pork products as a
source of zoonotic hepatitis E virus. Epidemiology and Infection 139,
1127–1144.