Detection of hepatitis E virus shedding in feces of pigs at different stages of production using reverse transcription-polymerase chain reaction.

Abstract

The aim of this study was to determine at which production stages hepatitis E virus (HEV) is shed by the highest number of pigs and to estimate the relative risk associated with each stage. For this purpose, 146 fecal samples of pigs from 21 farms were studied. In addition, 1 sample from the manure ditch and another sample of drinking water, collected directly from the trough located in the pen, were taken from 16 farms. HEV RNA was detected in fecal samples from 34 pigs (23.29%). The production stages in which most pigs excreted HEV were weaners (41.7%) and pigs in the first month of feeding (60%). The results of the statistical analysis showed that the principal significant risk stage in HEV shedding was the first month of feeding (odds ratio [OR] 19.5, 95% CI 3.59-106.07, P = 0.001) followed by the weaners stage (OR 9.3, 95% CI .78-48.42, P = 0.008). In 8 out of 16 farms tested (50%) HEV RNA was detected in raw manure and in the water trough of only 1. Detection of HEV in manure ditches raises the concern of how to deal with manure of swine origin, because it is used as soil fertilizer.

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Journal of Veterinary Diagnostic
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DOI: 10.1177/104063870601800506
2006 18: 462J VET Diagn Invest
Pérez-Gracia
Salceda Fernández-Barredo, Carolina Galiana, Angel García, Santiago Vega, María Teresa Gómez and María Teresa
Transcription-Polymerase Chain Reaction
Detection of Hepatitis E Virus Shedding in Feces of Pigs at Different Stages of Production Using Reverse


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J Vet Diagn Invest 18:462–465 (2006)
Detection of hepatitis E virus shedding in feces of pigs at different stages of production
using reverse transcription-polymerase chain reaction
Salceda Ferna ´ndez-Barredo, Carolina Galiana, Angel Garcı ´a, Santiago Vega, Marı ´a Teresa Go ´mez,
Marı ´a Teresa Pe ´rez-Gracia1
Abstract.
shed by the highest number of pigs and to estimate the relative risk associated with each stage. For this
purpose, 146 fecal samples of pigs from 21 farms were studied. In addition, 1 sample from the manure ditch
and another sample of drinking water, collected directly from the trough located in the pen, were taken from
16 farms. HEV RNA was detected in fecal samples from 34 pigs (23.29%). The production stages in which
most pigs excreted HEV were weaners (41.7%) and pigs in the first month of feeding (60%). The results of the
statistical analysis showed that the principal significant risk stage in HEV shedding was the first month of
feeding (odds ratio [OR] 19.5, 95% CI 3.59–106.07, P 5 0.001) followed by the weaners stage (OR 9.3, 95% CI
.78–48.42, P 5 0.008). In 8 out of 16 farms tested (50%) HEV RNA was detected in raw manure and in the
water trough of only 1. Detection of HEV in manure ditches raises the concern of how to deal with manure of
swine origin, because it is used as soil fertilizer.
The aim of this study was to determine at which production stages hepatitis E virus (HEV) is
Key words:Feces; hepatitis E virus; pigs; production stages; swine; zoonoses.
<!?show "fnote_aff1"$^!"content-markup(./author-grp[1]/aff|./author-grp[1]/dept-list)>
Hepatitis E virus (HEV) is the main causative agent of
non-A non-B enterically transmitted hepatitis with an acute
and self-limiting clinical presentation in humans.13,16
Mortality rates are low in the population, except for
pregnant women, especially in their third trimester, where
mortality rates increase dramatically and can reach 25%.
HEV was provisionally classified as belonging to the
Caliciviridae family from 1988 through 1998, but is now
classified in the separate genus Hepevirus in a new family
which has been named Hepeviridae. Although at least 4
major genotypes have been identified, only 1 serotype of
HEV is recognized.18
Epidemic and sporadic epidemiological forms of hepa-
titis E have been reported. The former is related to drinking
contaminated water in developing countries with poor
sanitary conditions.20The latter form has been reported
between epidemics in these areas, in patients with a history
of traveling to endemic areas, and in those in industrialized
countries who have not traveled abroad (autochthonous
hepatitis).14
Occurrence of autochthonous HEV infection in humans
has led to the screening of domestic animals for the
presence of the virus in developed countries, and a high
seroprevalence has been found, especially in swine farms. In
1997 the first swine strain HEV was isolated10and showed
a high percentage of homology with an autochthonous
human strain in USA (US-2). A year later, the experimental
From the Departamento de Atencio ´n Sanitaria, Salud Pu ´blica y
Sanidad Animal, Universidad Cardenal Herrera–CEU, Moncada
(Valencia), Spain.
1Corresponding Author: Marı ´a Teresa Pe ´rez-Gracia, Departa-
mento de Atencio ´n Sanitaria, Salud Pu ´blica y Sanidad Animal,
Facultad de Ciencias Experimentales y de la Salud, Universidad
Cardenal Herrera–CEU, Moncada 46113, Valencia, Spain.
teresa@uch.ceu.es
462
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infection of macaques with a swine HEV strain demon-
strated the ability of the virus to cross the species barrier,
but only mild clinical signs and/or biochemical alterations
were observed.11Several swine HEV isolates were sub-
sequently identified in pigs in Canada,22Japan,12Korea,4
New Zealand,5the Netherlands,19and the UK.2
The aim of this work was to study the prevalence of HEV
in pig feces in Spain and determine the production stages in
which HEV is shed by the highest number of pigs, in order
to estimate the relative risk associated with each stage. To
do this, 146 pigs were selected at random for the study from
21 farms. From January 2002 through August 2004 fecal
samples were collected directly from the rectum and placed
in sterile containers. Farms were of the following types: 5
farrow-to-weaning farms (farms that keep breeder sows,
piglets, and weaners, with piglets moved to nurseries from
weaning at 3 weeks up to 12 weeks), 11 grower-to-finish
farms (in these farms, 12-week-old weaners are bought and
fed until they reach 5 months), and finally 5 farrow-to-
finish farms (pigs of all production stages are kept on the
same farm). Pigs were divided into groups on the basis of
their production stages: 18 suckling piglets (from birth to
weaning); 24 weaners (pigs from weaning to 12 weeks old);
20, 20, and 28 pigs in the first, second, and third month of
feeding respectively; 32 breeder sows (adult females nursing
piglets); and 4 boars (adult males).
In addition, a sample from the raw manure ditch and
a sample of drinking water (obtained directly from the
trough in the pen) were taken from 16 farms. Samples from
the manure ditch and samples of feces were diluted with
phosphate-buffered saline (PBS) to 10% (w/v) suspensions.
These were centrifuged at 2000 3 g for 10 minutes at room
temperature; the supernatant was either used immediately
to extract RNA or kept frozen at 280uC until used.
RNA was extracted from 140 ml of each fecal, manure,
or water sample using a commercial kitafollowing the
manufacturer’s instructions. Two pairs of degenerated
oligonucleotide primers were used to amplify a 348-base-
pair (bp) fragment of open reading frame 2 (ORF-2) of
HEV using an RT-nested PCR.7These primers were based
on 18 human HEV sequences and the swine HEV prototype
strain from USA. A positive control from a naturally
infected pig (GenBank accesion number AY323506) was
included in each procedure. All assays were performed in
different places to avoid the possibility of cross-contami-
nation. The PCR products were separated by electropho-
resis in 2% agarose gel and were detected by staining with
ethidium bromide. Amplicons were purified and both
strands sequenced using a sequencing reaction kit.b
Sequences obtained in this study were deposited in the
GenBank database under accession numbers DQ093564,
DQ093566, DQ093567, DQ093568, DQ141118, DQ141119,
DQ141120, DQ141121, and DQ141127. A binary logistic
regression was performed to determine the effect of the
production stage on the prevalence of HEV in pig feces and
to calculate OR and their 95% CI. The fit of the final model
was assessed by the Hosmer-Lemeshow goodness-of-fit
test. Statistical analyses were performed using a statistical
analysis software package.cThe samples were considered
positive to the presence of HEV when a band of 348 bp was
seen in the agarose gel. HEV RNA was detected in fecal
samples of 34 pigs (23.3%) although none of them exhibited
clinical signs of infection.
Results according to the production stage of pigs are
shown in Table 1. Only 2 of the 3-week-old pigs out of 18
(11.1%) suckling piglets were shedding the virus. The
prevalence increased in weaners with a result of 41.7%,
reaching a maximum in the first month of feeding with
a prevalence of 60%. The minimum rate was observed in
the second month of feeding with 1 positive pig out of 20
(5%). Of the adult pigs, none of the 4 boars tested positive
for HEV RNA, but there was a prevalence of 21% in the
breeding sows. To determine the correlation between the
presence of HEV in feces and production stages, odds
ratios (ORs) and their corresponding 95% confidence
intervals (CIs) were calculated using binary logistic re-
gression analysis. A P value of less than 0.05 was
considered statistically significant. Table 1 shows the
results of the binary logistic regression analysis. In this
model, the first month of feeding (OR 19.5, 95% CI 3.59–
106.08, P 5 0.001) was recorded as the main significant risk
stage for shedding HEV, followed by the weaners stage
(OR 9.3, 95% CI 1.78–48.42, P 5 0.008). The Hosmer-
Lemeshow goodness-of-fit test showed a good fit for the
final model (x25 2.87, P 5 0.897).
At least 1 positive pig was detected on 8 of the 21 (38.1%)
farms tested (Table 2). HEV RNA was detected in manure
from 8 (50%) farms and in the water trough of 1 (6.25%)
Table 1.Detection of swine HEV RNA in pigs at different production stages.
Production stage
HEV in feces
Odds ratio (95% CI{)
P*Positive pigs / total (% positive)
Suckling piglets
Weaners
First month of feeding
Second month of feeding
Third month of feeding
Breeding sows
Boars
TOTAL
2 /18 (11.1%)
10/24 (41.7%)
12/20 (60.0%)
1/20 (5.0%)
2/28 (7.1%)
7/32 (21.9%)
0/4 (0%)
34/146 (23.3%)
1.6 (0.21–12.71)
9.3 (1.78–48.42)
19.5 (3.59–106.08)
0.7 (0.06–8.11)
1.0
3.6 (0.69–19.23)
0.00
0.644
0.008
0.001
0.764
—
0.128
0.999
{ CI 5 confidence interval.
* P values less than 0.05 were considered significant.
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farm out of 16. At least 1 pig was found to be positive and
HEV detected in manure in a total of 3 farms out of 8. The
same value was recorded for farms with at least 1 positive
pig, but negative for manure. The correlation between the
presence of HEV in pigs from a farm and the presence in
the manure ditch of the same farm was assessed using a x2
test, but no significant association was recorded. However,
a large number of samples would be necessary to confirm
these results. The percentage of infected pigs on farms
found to be negative for HEV in manure was 21.4% (12 out
of 56 pigs), whereas the percentage of farms found to be
positive for HEV in manure was 6.2% (2 out of 32).
To the authors’ knowledge, this is the first epidemiolog-
ical study in Europe to take each production stage of swine
into consideration. Total prevalence values (23.3%) ob-
tained in this work are similar to the few studies that have
tested feces from individual pigs, although the number of
animals in each case was fewer than in the present study. In
the UK, 26.19% of 12- to 15-week-old pigs were found to
be shedding HEV in feces on 2 farms studied.2In New
Zealand, 37.8% of 7- to 17-week-old pigs from 2 farms (45
pigs) were found to be shedding HEV in feces.5In the USA
25 (26.3%) 2- to 4-month-old pigs out of 95 were reported
to be shedding the virus.7
It takes between 2 and 3 weeks from initial infection to the
time when HEV is excreted in feces,11and the viral shedding
can last up to 7 weeks. Considering the fact that the
maximum prevalence recorded in this work was found in
the first monthof feeding (around 13 to16 weeks ofage),itis
reasonable to extrapolate that the majority of animals were
infected at 3 to 6 weeks of age from contact with the sows.
After an incubation period of 2 to 3 weeks, the virus would
have spread during the weaner stage and reached maximum
prevalenceinthefirstmonthof feeding.Thesecond andthird
months of feeding (just before slaughter) showed the lowest
prevalence values (5% and 7.1% respectively). This could be
due to the development of an effective immune response to
HEV at this stage. An interesting point is the relatively high
prevalence found in breeding sows (21.9%). It is known that
during pregnancy sows may shed some infectious organisms,
such as parasites and rotavirus, in higher titer.3,6,16This may
also be the case with HEV but, to the authors’ knowledge,
there is only 1 study of this stage: experimental infection of
pregnant gilts8found no clinical signs or documented
transmission in utero to the offspring, even though HEV
infection was successful and gilts shed HEV in feces. HEV
antibodies were detected in 10- to 61-day-old pigs born to
inoculated sows. As in other studies, no clinical signs were
recorded in the pigs sampled, so it would seem that the
infection is subclinical in pigs. Thus exposure in the form of
personal contact with subclinically infected animals could
result in HEV transmission from pigs to humans.
For practical purposes, the data on viral prevalence
according to production stages obtained in this study could
help to guide targeted sampling in the production stages
most at risk, in order to minimize the cost of testing on
a farm. In addition, it could also help prevent the
involuntary transmission by infected animals to an HEV-
free farm. There is no report of HEV RNA being detected
in manure ditches and water troughs on swine farms in
Europe. The values obtained in manure ditches were
surprisingly high, with 8 out of 16 farms testing positive
(50%) (Table 2). Only 1 water trough out of the 16 farms
was found to be positive (6.2%). The virus could have
reached the water trough though a pig’s snout that had
previously been in contact with a contaminated floor. No
correlation has been recorded between the presence of HEV
in pigs on a particular farm and the presence of HEV in the
manure ditch of that farm. Furthermore, the percentage of
infected pigs on farms found to be negative for HEV in
manure was much higher (21.4%) than that on farms found
to be positive for HEV in manure (6.2%). Thus, the analysis
of only 1 type of sample would decrease the sensitivity of
HEV detection on farms. The high presence of the virus in
manure ditches raises concern about its use as soil fertilizer.
A study of the viability of the virus15reported detectable
levels of HEV in sewage by RT-PCR over a 2-month
period. Thus it would be desirable to undertake studies to
determine the resistance of the virus in the environment in
order to evaluate the potential risk to humans, especially
when water is not tested for HEV. Correct treatment of
sewage or its routine screening and treatment of waste
materials may be needed to prevent HEV contamination of
waterways or areas around farms.
Several studies performed in Japan,12the UK,2and the
US10,11support the contentions that zoonotic infection
between pigs and humans may occur and that human and
swine HEV may coexist in nonendemic areas. Recent studies
suggest that autochthonous hepatitis E infection is under-
diagnosed in developed countries. A study in which sera
from 336 Spanish patients diagnosed for idiopathic acute
Table 2.
and from raw manure and trough for water in different types
of herds.*
Detection of swine HEV RNA from fecal samples
Farm
Type of
farm
Pigs positive/
tested (%)
Trough
water
Manure
ditch
1
2
3
4
5
6
7
8
9
F-W
F-W
F-F
F-F
G-F
G-F
G-F
G-F
G-F
F-F
F-W
G-F
G-F
G-F
G-F
G-F
G-F
F-W
F-F
F-W
F-F
0/8 (0)
8/8 (100)
0/8 (0)
0/8 (0)
0/4 (0)
1/4 (25)
1/4 (25)
1/4 (25)
0/4 (0)
3/12 (25)
0/8 (0)
0/4 (0)
0/4 (0)
0/4 (0)
0/4 (0)
0/4 (0)
0/4 (0)
1/6 (16.6)
12/19 (63.2)
0/6 (0)
7/19 (36.8)
34/146 (23.3)
NEG
NEG
N/A
NEG
NEG
NEG
NEG
NEG
NEG
NEG
NEG
NEG
POS
NEG
NEG
NEG
NEG
N/A
N/A
N/A
N/A
1/16 (6.2)
NEG
NEG
N/A
NEG
POS
POS
NEG
POS
NEG
NEG
NEG
NEG
POS
POS
POS
POS
POS
N/A
N/A
N/A
N/A
8/16 (50)
10
11
12
13
14
15
16
17
18
19
20
21
Total
* F-W 5 farrow-to-weaning; F-F 5 farrow-to-finish; G-F 5
grower-to-finish; N/A 5 not available.
464
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hepatitis was reevaluated14found seroprevalence of 8.9%
using anti-HEV IgM. Other studies carried out in several
European countries such as Austria,21England,2France,9
and Italy23have found autochthonous cases with a high
percentage of nucleotide similarity to porcine strains from
thesamegeographic area.Insummary,thisworkcouldserve
to highlight the production stages in which pigs are in most
danger of infecting other pigs. The presence of swine manure
indicates the potential for spread to humans through contact
with contaminated crops or in personnel that handle swine
manure and spread this waste on agricultural fields.
Acknowledgements.
This work was supported by grants
from Cardenal Herrera–CEU University (PRUCH 04/8),
Escuela Valenciana de Estudios parala Salud (053/2005),
and Consellerı ´a de Empresa, Universidad y Ciencia de la
Generalitat Valenciana (GV05/132). S. Ferna ´ndez-Barredo
is a grant holder of Cardenal Herrera–CEU University. We
are grateful to Ms. Margot Ovenden for the assistance in
translating the manuscript, and Ms. Paloma Botella has
kindly helped in statistical analysis of the data.
Sources and manufacturers
a. QIampViral RNA Kit, Qiagen, Valencia, CA.
b. ABIPRISM Dye Terminator Cycle Sequencing Ready Re-
action kit, Perkin-Elmer, Applied Biosystems, Foster City,
CA.
c. SPSS software package (version 12.0). SPSS Inc., Chicago,
IL.
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