Bull Vet Inst Pulawy 59, 185-190, 2015
DOI: 10.1515/bvip-2015-0028
Occurrence of Aujeszky’s disease virus
single-reactor pigs
in Poland
Andrzej Lipowski, Zygmunt Pejsak
Department of Swine Diseases,
National Veterinary Research Institute, 24-100 Pulawy, Poland
[email protected]
Received: January, 14 2015
Accepted: June, 8 2015
Abstract
The aim of the study was to analyse the prevalence of single-reactor (SR) pigs in Poland, to attempt an explanation for this
phenomenon, and to assess whether the occurrence of SR pigs could create problems for a successful Aujeszky’s disease virus
(ADV) eradication programme in Poland. A total of 6494 ADV gE antibody positive/doubtful sera were retested by gB ELISA
and subsequently by virus neutralisation test (VNT) to confirm the results of the glycoprotein E (gE) ELISA. Amongst the serum
samples tested, 5.9% could be classified as being taken from SR pigs, as was shown by gE ELISA positive/doubtful results,
which were not confirmed by negative findings in gB ELISA and VNT. It means that the observed SR phenomenon was due to
a false positive/doubtful reaction in gE ELISA. This finding was strongly supported by the fact that the serum samples were
taken from the animals from herds without any previous or subsequent history of Aujeszky’s disease. The low percentage of SR
pigs does not seem to create a big obstacle to a successful ADV eradication programme in Poland.
Keywords: pigs, Aujeszky’s disease, Aujeszky’s disease virus, single-reactor, Poland.
Introduction
Aujeszky’s disease (AD), caused by Suid
herpesvirus 1 (4) which is also called Aujeszky’s
disease virus (ADV) or pseudorabies virus (PrV), is
still responsible for heavy economic losses in the pig
industry all over the world (16). Besides the direct or
indirect effects caused by this disease itself, the
restrictions or even ban on international pig
movement and trade mar the industry significantly (3,
5, 10, 12). Such restrictions have been in force in the
European Community (EC) since 1993 and have
spurred many EC member states to start AD
eradication programmes (11). One of the very
important elements of these programmes was
intensively conducted serological surveillance that
allowed estimation of ADV seroprevalence in a region
or a state (13). Moreover, the results of this
serological testing were used as a basis for
administrative decisions in regard to the chosen
eradication method, i.e. by using a vaccination–
eradication method or by culling infected herds.
Serological monitoring, especially performed in
the final stage of an eradication programme, revealed
the presence in many countries of single-reactor (SR)
pigs, i.e. individual seropositive pigs in ADV free
farms (1, 7, 8, 20). The phenomenon of single
seropositive animals concerns serum samples tested
by ADV whole antigen ELISA (1), glycoprotein B
(gB) ELISA (20), or by glycoprotein E (gE; formerly
gpI or gI) ELISA (7, 8).
The introduction of ADV into a pig herd usually
results in more or less rapid spread of the virus (7)
and a high percentage of pigs become seropositive. As
a consequence of ADV replication, a latent infection
in the central nervous system is established, which,
from a practical point of view, is impossible to detect
by traditional virological methods (16). Stress
situations (e.g. pig movement, feeding system change,
environment change etc.) can result in virus
reactivation and dissemination throughout the body,
followed by an immunological reaction detected by
serological tests, e.g. ELISA. Such latently infected
pigs might be detected from time to time, creating
© 2015 A. Lipowski et al. This is an open access article distributed under the Creative Commons AttributionNonCommercial-NoDerivs license (http://creativecommons.org/licenses/by-nc-nd/3.0/)
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a serious problem during an ADV eradication
programme (14).
In addition to the mentioned seroconversion
mechanism in the final stage of the ADV eradication
programme in many countries (1, 7, 8, 20), the
unexpected problem of SR pigs was discovered that
rendered serological result interpretation difficult and
has ramifications for administrative decisions
concerning herds with SR pigs. Herds having SR
animals present a dilemma for both the producer and
regulatory officials since the AD status of the herd is
unclear and choosing the appropriate course of action
is difficult (1).
In Poland in 2009 during serological testing, 10%
of pig sera were recognised as sera from SR pigs (19).
That was the reason to undertake the present
investigation.
The aims of the study were to analyse the
prevalence of SR pigs in Poland, to attempt an
explanation for this phenomenon, and to assess
whether the occurrence of SR pigs could create
problems and became a real threat to a successful
ADV eradication programme in Poland, especially in
its final stage.
Material and Methods
A total of 6494 swine sera from the whole
territory of Poland were delivered between 2010 and
2013 by regional Veterinary Diagnostic Laboratories
(VDL) of the Veterinary Inspectorate to the National
Reference Laboratory for Aujeszky’s disease
(Department of Swine Diseases, National Veterinary
Research Institute, Pulawy, Poland) for verification of
ADV serological status.
Serological examination. Basic examination was
performed with the use of ELISA and virus
neutralisation test (VNT). ELISA Pseudorabies Virus
gpI Antibody Test Kit (IDEXX PRV/ADV gI, IDEXX
Laboratories Inc., USA) and Pseudorabies Virus gB
Antibody Test Kit (IDEXX PRV/ADV gB, IDEXX
Laboratories Inc., USA) were both performed
according to the manufacturer’s instructions. Samples
positive and/or doubtful in IDEXX PRV/ADV gI and
negative in IDEXX PRV/ADV gB were examined for
ADV antibody by means of a virus neutralisation test
(VNT). Briefly, the sera were diluted from 1:2 up to
1:256 and then incubated for 1 h at 37 ±2 oC with the
NIA-3 strain of ADV (15) at a dose of 100
TCID50/50µL and supplemented with SK-6
continuous cell line (9). During 5 d of incubation in a
CO2 incubator (5 ± 1% CO 2) at 37 ± 2 oC, observations
for cytopathic effect were performed.
Additional examination was conducted using
ELISA PrioCHECK PRV gB (Prionics Lelystad B.V.,
the Netherlands) for verification of IDEXX
PRV/ADV gB results, performed according to the
manufacturer’s instructions.
Out of 386 positive and/or doubtful samples in
the gE ELISA, only 357 sera were verified by
PrioCHECK PRV gB ELISA in the additional
examination. This limitation upon the number of
tested samples resulted from insufficient sera volume
due to their previous use in the gE/gB ELISAs and
VNT.
Results
The results of the basic serological examination
(IDEXX gI/gB and VNT) are shown in Table 1 and
the results of the additional examination are shown in
Table 2. Single-reactor pig prevalence is shown in
Fig. 1.
The basic examination data indicate that out of
6494 samples, 386 sera which were positive/doubtful
in gE ELISA gave negative results in gB ELISA. Of
these, 380 (5.9%) were negative in VNT and
recognised as false positive, i.e. taken from SR to
ADV pigs. Six sera were cytotoxic, so it was not
possible to take the readings of the results of VNT.
Single-reactor to ADV pigs were detected in all
voivodships examined (Fig. 1) except Pomorskie
(PM). The highest number of samples originated from
Wielkopolskie (WP) Voivodship (3301 samples) and
among them 134 (4.1%) were from SR pigs. The
lowest number of sera were received from Lubuskie
(LB) Voivodship (6 samples) and only 1 sample
(16.7%) was regarded as coming from an SR pig.
The percentage of SR pigs ranged from 1.8% in
Podkarpackie (PK) Voivodship to 50% in ZachodnioPomorskie (ZP) Voivodship, with an average of 5.9%
(Table 1) within the whole period of examination. In
samples originating from PM Voivodship no SR
animals were detected.
The additional examination data (Table 2) report
that out of 6465 samples, 357 sera were tested using
PrioCHECK PRV gB ELISA. Of these, 348 (5.4%)
yielded the same results as those tested by the basic
examination. It means that the results of the
aforementioned test confirmed that the sera
recognised as false positives were taken from SR to
ADV pigs. Nine samples were positive in additional
gB ELISA and 6 of these were cytotoxic in VNT. The
remaining 3 samples were negative in IDEXX
PRV/ADV gB ELISA and VNT.
Discussion
In Poland, the AD eradication programme started
in 2008 and engendered intensive serological testing
(17, 18).
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Table 1. Results of basic serological examination using IDEXX PRV/ADV gI and IDEXX PRV/ADV gB ELISA kits and VNT for ADV singlereactor pigs
False gE ELISA
positive/doubtful results
Number of sera
Voivodship (abbr.)
tested by
gE and gB
ELISA
gE ELISA positive/doubtful
and gB ELISA negative
tested by VNT
SR pigs
number
SR pigs
(%)
Dolnośląskie (DS)
Kujawsko-Pomorskie (KP)
Lubelskie (LU)
Lubuskie (LB)
Łódzkie (LD)
Małopolskie (MA)
Mazowieckie (MZ)
Opolskie (OP)
12
833
547
6
402
245
118
339
5
22
83
1
41
8
11
10
5
22
83
1
41
8
11
10
5
22
83
1
41
8
11
10
Podkarpackie (PK)
216
4
4
4
1.8
Podlaskie (PD)
Pomorskie (PM)
Śląskie (SL)
Świętokrzyskie (SK)
Warmińsko-Mazurskie (WN)
Wielkopolskie (WP)
Zachodnio-Pomorskie (ZP)
65
77
149
118
54
3301
12
25
13
15
4
138
6
25
13
15
4
138
6
24a
13
14b
4
134c
6
36.9
8.7
11.9
7.4
4.1
50.0
Total
6494
386
386
380
5.9
41.7
2.6
15.2
16.7
10.2
3.3
9.3
2.9
a)1 sample was cytotoxic
b)1 sample was cytotoxic
c)4 samples were cytotoxic
Table 2. Results of additional examination using PrioCHECK PRV gB ELISA kit for ADV single-reactor pigs
Number of sera
Voivodship (abbr.)
False gE ELISA
positive/doubtful results
Positive results in
PrioCHECK PRV gB
number
percentage
available for testing
tested by
PrioCHECK PRV
gB
SR pigs
number
SR pigs (%)
Dolnośląskie (DS)
Kujawsko-Pomorskie (KP)
Lubelskie (LU)
Lubuskie (LB)
Łódzkie (LD)
Małopolskie (MA)
Mazowieckie (MZ)
Opolskie (OP)
9
833
545
6
401
242
118
338
2
22
81
1
40
5
11
9
2
22
81
1
37
6
11
9
22.2
2.6
14.9
16.7
9.2
2.5
9.3
2.7
3a
-
0.7
-
Podkarpackie (PK)
216
4
4
1.9
-
-
b
Podlaskie (PD)
Pomorskie (PM)
Śląskie (SL)
Świętokrzyskie (SK)
Warmińsko-Mazurskie (WN)
Wielkopolskie (WP)
Zachodnio-Pomorskie (ZP)
63
77
149
118
54
3284
12
23
13
15
4
121
6
22
13
14
4
117
6
35.0
8.7
11.9
7.4
3.6
50.0
1
1c
4d
-
1.6
0.8
0.1
-
Total
6465
357
348
5.4
9
0.1
a)3 samples were negative in IDEXX PRV/ADV gB and in VNT
b)1 sample was cytotoxic
c)1 sample was cytotoxic
d)4 samples were cytotoxic
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Fig. 1. Prevalence of single-reactor pigs to ADV in Poland
Full names of all voivodships are included in Tables 1 and 2
Serological tests (gE ELISA) were performed in
all 16 regional VDLs to recognise the epidemiological
situation of AD in all pig holdings (i.e. backyards,
breeding farms, farrow-to-finish farms, and fattening
farms) and in counties, voivodships, and the whole
country. All results obtained in VDLs were analysed
and in case of doubtful samples or suspected AD
(e.g. a single gE positive pig), the samples were sent to
the National Reference Laboratory for Aujeszky’s
Disease at the Institute for verification as mandated
under the programme. Here ELISA kits to detect
antibodies to gE and gB of ADV simultaneously were
used. Three possible interpretations of the results were
taken into account:
a) gE positive, gB positive - the pig is infected
with ADV;
b) gE negative, gB positive - the pig is not
infected but vaccinated with gE negative (gE
deleted) vaccine;
c) gE positive/doubtful, gB negative - the pig is an
SR animal (the gE result is recognised as false
positive/doubtful).
The study detected some pigs with a result
bringing interpretation c) mentioned above. Such
a serological reaction was more often observed in sows
older than 18 months of age than in other pigs.
The presented study is unique because of its
complexity, and because the investigation of SR pig
prevalence has been performed only in a very few
laboratories in the world, so the literature concerning
this problem is very scarce. Nowadays, in countries
where AD has already been eradicated, including most
European Union member states, such studies are not
undertaken. Nevertheless, in many countries (e.g. USA,
Great Britain, the Netherlands, Norway, Germany, and
Sweden) detailed analysis of ADV serological
monitoring results has revealed quite a high percentage
of pig holdings with SR pigs. In most of these holdings
intensive serological testing did not detect other single
reactors to ADV (1, 7, 8, 20).
In Sweden, 1300 SR animals were detected during
serological testing with the gB ELISA, performed on
480 000 pigs from 8900 farms (20). Most of these pigs
(99%) reacted negatively in the gE ELISA. What was
interesting was that no AD symptoms nor virus
transmission were observed in these farms (20). To
elucidate these findings, an experiment was set up
using a group of SR animals and negative control ones.
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Of the laboratory methods applied, only the most
sensitive one, i.e. PCR, allowed the detection of some
amplicons with very high homology with the
corresponding sequences of the gB, gE, and gD genes
of ADV in the organs of SR animals. On the other
hand, no latency transcripts were detected in the organs
of SR pigs. All the results indicated that the SR animals
used in the experiment were not infected with ADV at
all. Taking into account that vaccination was not
performed in Sweden, that SR pigs occur not only in
the south but also in the north of Scandinavia, and that
ADV reactivation was not observed under natural
conditions nor after experimental immunosuppression,
the authors concluded that the SR phenomenon could
not be considered a typical ADV latent infection (20).
The results of the mentioned experiments indicate that
certain herpesviral genomic sequences could exist in
healthy uninfected pigs (20).
A quite different situation was found by American
epidemiologists (1). In Minnesota, ADV serological
monitoring conducted in 173 of 16 500 pig herds
revealed SR animals in 30 herds. The unexpected
results of this monitoring prompted the undertaking of
a detailed analysis of the situation based on laboratory
investigation of SR pigs. To do this, 27 SR animals
were selected from 19 out of the 30 herds. Several
laboratory tests were conducted and positive results
were obtained only in 4 of the 27 SR pigs. In 4 out of
the 19 analysed herds, outbreaks of AD occurred 20–30
months after the detection of SR animals, but their
source was never determined (1). Irrespective of the
outbreak cause, 23 SR pigs from which no virus was
isolated were false positives in serological tests, so they
represent true SR animals.
The samples from Wielkopolskie (WP)
Voivodship seem to confirm the theory about the lack
of correlation between the epidemiological situation
regarding AD and the percentage of SR animals
detected in the region. This study sent 3301 serum
samples from WP Voivodship for confirmatory testing.
The high number of samples was due to the fact that
this region has the highest pig density and production
intensity in Poland. As a result, a higher ADV infection
rate was detected in this voivodship than in other ones
(22). But the results of the presented study show that
the percentage of SR pigs in WP Voivodship was 4.1%
and thereby lower than in the whole of Poland (5.9%).
A similarly low percentage of SR animals (2.6%) was
detected in Kujawsko-Pomorskie (KP) Voivodship,
which is the second Polish pig producing region.
Contrastingly, in voivodships with low pig density
population, i.e. in Dolnośląskie (DS) and ZachodnioPomorskie (ZP), SR pigs accounted for 41.7% and 50%
respectively. However, all the results should be
assessed carefully considering the number of samples
sent and tested.
Analysing the reasons for the SR phenomenon,
a wide range of scientifically proven possibilities
189
should be taken into account. To reach an explanation,
many different methods have been applied of virus
isolation from immunosuppressed pigs in which
reactivation of ADV and the presence of infectious
virus was supposed to have been detected (1, 3, 20).
Also to detect ADV, an immunofluorescence test (1),
an in situ hybridisation test (3, 20), and PCR (3, 6, 7, 8,
20) were used in addition.
Based on the different results of the
aforementioned studies, the seropositive status of SR
pigs could be explained as follows (1, 20):
1. a false positive reaction in the serological assay;
2. the first animal infected in the herd;
3. the last animal infected in the herd;
4. the only animal infected in the herd;
5. an animal infected with an ADV strain of very
low virulence; or
6. the occurrence of certain ADV-related genes in
an apparently uninfected animal.
It may be assumed from these postulates that the
detection of SR animals in Poland was due to false
positive/doubtful results of gE ELISA. In general, such
results could be explained by the high sensitivity and
low specificity of the ELISA kits used. Furthermore,
the ELISA results could be influenced by the method of
bleeding and transportation to the laboratory, serum
separation method, and period between sampling and
testing the serum. During the study all these potential
causes were excluded.
In conclusion, the present study shows 5.9% of the
6494 samples sent by VDLs to the Institute for
confirmatory testing to be samples deriving from SR
pigs. This was proved by testing questionable gE
ELISA positive/doubtful samples by gB ELISA and
VNT with negative results. It means that the observed
phenomenon of SR was due to the false positive and/or
doubtful reaction in gE ELISAs. This finding is
strongly supported by the fact that the samples tested
were taken from animals from herds without previous
or subsequent history of AD. Furthermore, the low
percentage of truly false positive/doubtful gE ELISA
results indicate that ADV SR pigs do not seem to create
a big obstacle to a successful ADV eradication
programme in Poland, especially in its final stage. The
practical solution to this problem was the amendment
of the Ministry Council regulation concerning the
national AD eradication programme, which mandated
that all the SR pigs have to be simply eliminated from
the herds by culling.
Conflict of Interests Statement: The authors declare
that there is no conflict of interest regarding the
publication of this article.
Financial Disclosure Statement: This work was
supported by the National Science Centre (grant no.
N N308 577739).
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Acknowledgements: The authors would like to thank
Mrs Renata Wydra and Mrs Barbara Małek for their
skilled technical assistance.
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