pathogens
Article
Cross-Sectional Study on the Prevalence of PCV Types 2 and 3
DNA in Suckling Piglets Compared to Grow–Finish Pigs in
Downstream Production
Matthias Eddicks 1, *, Roland Maurer 1 , Pauline Deffner 1 , Lina Eddicks 2 , Wolfgang Sipos 3 , Sven Reese 4 ,
Vojislav Cvjetković 5 , Roman Krejci 6 , Tanja Opriessnig 7,8 , Mathias Ritzmann 1 and Robert Fux 9
1
2
3
4
5
6
7
8
Citation: Eddicks, M.; Maurer, R.;
9
Deffner, P.; Eddicks, L.; Sipos, W.;
Reese, S.; Cvjetković, V.; Krejci, R.;
*
Clinic for Swine, Centre for Clinical Veterinary Medicine, Ludwig-Maximilians-Universität München, 85764
Oberschleissheim, Germany; r.maurer@med.vetmed.uni-muenchen.de (R.M.);
p.deffner@med.vetmed.uni-muenchen.de (P.D.); m.ritzmann@lmu.de (M.R.)
Institute of Veterinary Pathology, Centre for Clinical Veterinary Medicine, Ludwig-Maximilians-Universität
München, 80539 Munich, Germany; lina.eddicks@patho.vetmed.uni-muenchen.de
Clinical Department for Farm Animals and Herd Management, University of Veterinary Medicine Vienna,
1210 Vienna, Austria; wolfgang.sipos@vetmeduni.ac.at
Institute for Anatomy, Histology and Embryology, Ludwig-Maximilians-Universität München,
80539 Munich, Germany; s.reese@anat.vetmed.uni-muenchen.de
Ceva Tiergesundheit GmbH, 40472 Duesseldorf, Germany; vojislav.cvjetkovic@ceva.com
Ceva Santé Animale, 33500 Libourne, France; roman.krejci@ceva.com
The Roslin Institute, The Royal (Dick) School of Veterinary Studies, University of Edinburgh, Easter Bush,
Midlothian EH25 9RG, UK; tanja.opriessnig@roslin.ed.ac.uk
Department of Veterinary Diagnostic and Production Animal Medicine, College of Veterinary Medicine, Iowa
State University, Ames, IA 50011, USA
Division of Virology, Department of Veterinary Sciences, Ludwig-Maximilians-Universität München,
80539 Munich, Germany; robert.fux@lmu.de
Correspondence: m.eddicks@lmu.de
Opriessnig, T.; Ritzmann, M.; et al.
Cross-Sectional Study on the
Prevalence of PCV Types 2 and 3
DNA in Suckling Piglets Compared
to Grow–Finish Pigs in Downstream
Production. Pathogens 2022, 11, 671.
https://doi.org/10.3390/
pathogens11060671
Academic Editors: Giovanni Franzo
and Michele Drigo
Received: 5 May 2022
Accepted: 5 June 2022
Published: 10 June 2022
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
Abstract: Vertical transmission is a consistently discussed pathway of porcine circovirus type 2
(PCV2) and porcine circovirus type 3 (PCV3) transmission in pigs. To evaluate the presence of PCV2
and PCV3 in piglets, we collected tissue samples from 185 piglets that were crushed within the first
week of life from 16 farms located in Germany and Austria. Pooled samples consisting of thymus,
inguinal lymph node, myocardium, lung and spleen were examined for PCV2 and PCV3 by qPCR.
Furthermore, oral fluid samples (OFS) from grow–finish pigs were collected and examined the same
way. In piglets, PCV2 was highly prevalent (litters: 69.4%; piglets: 61.6%), whereas PCV3 prevalence
was low (litters: 13.4%; piglets: 13.0%). In total, 72.6% and 67.2% of all collected OFS were PCV2
or PCV3 positive, respectively. Sow vaccination against PCV2 was identified as a protective factor
concerning PCV2 in piglets (OR: 0.279; CI: 0.134–0.578; p < 0.001), whereas the porcine reproductive
and respiratory syndrome virus (PRRSV) vaccination of sows was identified as a protective factor
concerning PCV3 in piglets (OR: 0.252 CI: 0.104–0.610; p = 0.002). Our results show that PCV2, but
not PCV3, is ubiquitous in suckling piglets and that early PCV3 infections might be modulated
by PRRSV–PCV3 interaction. However, the ubiquitous nature of both viruses in older pigs could
be confirmed.
iations.
Keywords: porcine circoviruses; oral fluids; suckling piglets; tissue samples
Copyright: © 2022 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
1. Introduction
Porcine circovirus type 2 (PCV2) is an economically and health-relevant pathogen in
the domestic pig population that is associated with several disease syndromes (porcine
circovirus diseases; PCVD) affecting growing pigs as well as breeding animals [1]. In breeding herds, piglets can be infected with PCV2 horizontally via shedding of infectious virus
particles by the dam or neighboring sows [2,3], or vertically via diaplacental intrauterine
Pathogens 2022, 11, 671. https://doi.org/10.3390/pathogens11060671
https://www.mdpi.com/journal/pathogens
Pathogens 2022, 11, 671
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infection [3,4]. The relevance of the intrauterine way of transmission was demonstrated
in several experimental and field studies over the last two decades. Whereas results from
North America and Brazil showed high amounts of PCV2 DNA-positive sera from newborn
piglets [5–7], results from Europe exhibit contrary results [8,9], which may be due to the
differences of PCV2 infection dynamics and PCV2 vaccination status at the same time of
sample collection. However, it is undisputed that the diaplacental transmission of PCV2
plays a crucial role in the spread of PCV2 within the domestic pig population and, among
others, could lead to latent PCV2 infections [10]. Recent study results from a subclinically PCV2-infected German multiplier herd indicate the relevance of PCV2 viremia in
the early stage of gestation for this way of transmission, particularly concerning young
sows [11]. Furthermore, Dvorak et al. [5] demonstrated that besides the diaplacental route
of transmission, the early infection of piglets can also occur due to environmental PCV2
contamination in the farrowing pen. The chance for piglets to become infected with PCV2
in the early stages of life was further elucidated by a study on in vivo-derived porcine
morulae- and blastocyst-stage embryos [12]. Specifically, it was shown in an experimental
study design that zona pellucida-free morulae and blastocysts are susceptible to PCV2
infection. In a Spanish field study, the positive effects of PCV2 vaccination in a subclinically
infected breeding herd included improved pig vitality and higher numbers of live-born
piglets, which further supports the hypothesis that PCV2 infections during gestation occur
regularly [13]. Taken together, there is a vast number of possibilities for piglets to become
infected with PCV2 in the early stages of life.
Concerning porcine circovirus type 3 (PCV3), similar to PCV2, associations of this virus
with different diseases or clinical outcomes in pigs have been proposed, including porcine
dermatitis and nephropathy syndrome (PDNS) [14], porcine respiratory diseases complex
(PRDC) [15], congenital tremor [16] or reproductive disorders [17,18]. Experimental proof of
these associations is still lacking for PCV3, however, the detection of PCV3 DNA in aborted
fetuses [17,18] indicates a diaplacental route of infection and subsequent PCV3 transmission
to naïve littermates in the early stages of life. Furthermore, Kedkovid et al. [19] demonstrated that in analogy to PCV2 [20], PCV3 DNA can also be detected in sow colostrum.
Based on the current knowledge of early PCV2 and PCV3 infections, we hypothesized
that the horizontal transmission of PCV3 to littermates is a frequent event in early life, but
also in later stages of production. The present study was designed to evaluate the detection
rates of the two virus species in suckling piglets under field conditions. Specifically, tissue
samples from piglets that were crushed by their mothers within the first week of life were
collected and analyzed, while oral fluid samples (OFS) were collected from older pigs.
Utilizing crushed piglets enabled a purposeful sampling of different tissues with respect
to “the three Rs” (Replacement, Refinement and Reduction) desired for scientific working
with animals to avoid animal experiments and to limit the number of animals and their
suffering, as published in Directive 2010/63/EU of the European Parliament and of the
Council of 22 September 2010 on the protection of animals used for scientific purposes.
2. Results
2.1. Presence of PCV2 and PCV3 DNA in Suckling Piglets
PCV2 DNA-positive suckling piglets were present on 15 of 16 farms (Germany 13/13,
Austria 2/3). In total, 64.9% (95% CI: 56.6–73.1%) of all litters and 61.6% (95% CI: 55.1–68.6%)
of the individual piglets were PCV2 DNA-positive, respectively. PCV3 was detected in
tissue samples from 9/16 farms (8/13 Germany; 1/3 Austria). In 13.4% (95% CI: 7.9–19.5%)
of all litters and 13.0% (95% CI: 8.1–17.8%) of the individual piglets, PCV3 DNA was present.
Dual PCV2 and PCV3 infection in the tissue samples of the individual suckling piglets was
detected in 7.0% (95% CI: 3.8–10.8%). Chi2 testing revealed that significantly more litters
and significantly more piglets were PCV2- than PCV3-positive (p < 0.001). The number of
PCV2 and PCV3 DNA-positive litters, piglets and OFS for each individual farm is shown
in Table 1.
Pathogens 2022, 11, 671
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Table 1. PCV2 and PCV3 DNA-positive litters, piglets, and OFS on each farm (n/%).
Suckling Piglets
(Up to 1 Week of Age)
Farm
Tissue Pools by Litters
Grow–Finish Pigs
(6–20 Weeks of Age)
Tissue Pools for Each Piglet
Oral Fluids
PCV2
% (n)
PCV3
% (n)
PCV2
% (n)
PCV3
% (n)
PCV2
%(n)
PCV3
% (n)
1
41.7%
(5/12)
8.3%
(1/12)
38.5%
(5/13)
7.7%
(1/13)
100.0%
(32/32)
65.6%
(21/32)
2
68.8%
(11/16)
6.3%
(1/16)
66.7%
(12/18)
5.6%
(1/18)
60.0%
(21/35)
65.7%
(23/35)
3
75.0%
(6/8)
37.5%
(3/8)
77.8%
(7/9)
33.3%
(3/9)
63.9%
(23/36)
30.6%
(11/36)
4
87.5%
(14/16)
0.0%
(0/16)
78.6%
(22/28)
0.0%
(0/28)
63.9%
(23/36)
22.2%
(8/36)
5
42.9%
(3/7)
28.6%
(2/7)
44.4%
(4/9)
22.2%
(2/9)
62.2%
(23/37)
100.0%
(37/37)
6
75.0%
(3/4)
0.0%
(0/4)
75.0%
(6/8)
0.0%
(0/8)
53.1%
(17/32)
87.5%
(28/32)
7
100.0%
(9/9)
0.0%
(0/9)
92.3%
(12/13)
0.0%
(0/13)
42.9%
(15/35)
62.9%
(22/35)
8
100.0%
(8/8)
12.5%
(1/8)
75.0%
(12/16)
6.3%
(1/16)
96.9%
(31/32)
81.3%
(26/32)
9
89.5%
(17/19)
5.3%
(1/19)
85.7%
(18/21)
4.8%
(1/21)
29.4%
(10/34)
73.5%
(25/34)
10
80.0%
(4/5)
100.0%
(5/5)
72.7%
(8/11)
100.0%
(11/11)
57.1%
(16/28)
92.9%
(26/28)
11
50.0%
(1/2)
0.0%
(0/2)
50.0%
(1/2)
0.0%
(0/2)
100.0%
(36/36)
61.1%
(22/36)
12
50.0%
(3/6)
50.0%
(3/6)
36.4%
(4/11)
27.3%
(3/11)
100.0%
(38/38)
94.7%
(36/38)
13
11.1%
(1/9)
11.1%
(1/9)
8.3%
(1/12)
8.3%
(1/12)
100.0%
(24/24)
95.8%
(23/24)
14
33.3%
(1/3)
0.0%
(0/3)
33.3%
(1/3)
0.0%
(0/3)
40.0%
(8/20)
0.0%
(0/20)
15
0.0%
(0/7)
0.0%
(0/7)
0.0%
(0/7)
0.0%
(0/7)
87.0%
(20/23)
39.1%
(9/23)
16
33.3%
(1/3)
0.0%
(0/3)
25.0%
(1/4)
0.0%
(0/4)
100.0%
(37/37)
78.4%
(29/37)
Total
64.9%
(87/134)
13.4%
(18/134)
61.6%
(114/185)
13.0%
(24/185)
72.6%
(374/515)
67.2%
(346/515)
2.2. Qualitative Evaluation
Dichotomous variables were first checked for significant associations using the chi2
test. Significantly associated factors (p ≤ 0.05) were subsequently included in a binary
logistic regression. The results concerning PCV2 and PCV3 DNA-positive tissue pools are
shown in Tables 2–4. In brief, PCV2 sow vaccination was a protective factor concerning the
presence of PCV2 DNA in piglets while sow vaccination against the porcine reproductive
and respiratory syndrome virus (PRRSV) was identified as a protective factor for the
presence of PCV3 DNA in piglets. On-farm replacement of gilts tended to be negatively
associated with numbers of PCV2 DNA-positive piglets.
Pathogens 2022, 11, 671
4 of 10
Table 2. Results of chi2 test and binary logistic regression of dichotomous variables for PCV2 (only
significant results shown here).
Independent
Variable
Dependent
Variable
p-Value
Chi2 Test
p-Value
Binary Logistic
Regression
OR
Upper CI
Lower CI
<0.001
0.001
0.279
0.134
0.578
0.004
0.464
-
-
-
0.007
0.056
-
-
-
PCV2
sow vaccination
PCV2 DNApositive piglet
Mycoplasma
hyopneumoniae sow
vaccination
Own replacement gilts
Table 3. Results of chi2 -test and binary logistic regression of dichotomous variables for PCV3 (only
significant results shown here).
Independent
Variable
Dependent
Variable
Porcine reproductive and
respiratory syndrome virus
sow vaccination
Actinobacillus
pleuropneumoniae sow
vaccination
p Value
Chi2 Test
p-Value
Binary Logistic
Regression
OR
Upper CI
Lower CI
0.001
0.002
0.252
0.104
0.610
0.048
0.184
-
-
-
PCV3 DNApositive piglet
Table 4. Percentage and number of PCV2 or PCV3 DNA-positive piglets with respect to PCV2 or
PRRSV sow vaccination.
PCV2 Sow
Vaccination
PCV2 DNA-Positive Piglets % (n)
PRRSV Sow Vaccination
PCV3 DNA-Positive Piglets % (n)
Yes
37.5% (CI: 22.2–53.7%)
(15/40)
Yes
8.1% (CI: 3.7–13.2%)
(11/135)
No
68.3% (CI: 60.8–75.7%)
(99/145)
No
26.0% (CI: 14.9–38.5%)
(13/50)
2.3. Quantitative Evaluation in Tissue
To assess whether the variables from the binary logistic regression model also affect
the viral load in the samples, we conducted a generalized mixed model that included the
same independent variables as the regression and additionally considered double infection
with PCV2 and PCV3. For this examination, only PCV2, PCV3 and PCV2 and PCV3 DNApositive samples were included. This examination revealed that neither for PCV2 nor for
PCV3 do any of the factors significantly influence the Cq value in the tissue samples.
The further statistical analysis included examinations concerning the relationship
(Spearman’s rho) between the quantitative outcome (Cq values) of the PCRs for tissue
sample pools for PCV2 and PCV3 and the factors “age of the sampled piglet”, “bodyweight
of the sampled piglet”, and “parity of the corresponding dam” or “life-borne piglets in the
corresponding litter”, “dead-borne piglets in the corresponding litter” and “bodyweight of
the sampled pig”. These examinations revealed that the viral load of PCV3 in the tissue
pools increased with the increasing age of the sampled piglets (rs : −0.413; p = 0.045). In
the generalized mixed model, this observation turned to a tendency (p = 0.058). No further
significant correlation was observed. The Cq values for the tissue pools under consideration
of the age of the sampled piglet are shown in Figure 1.
Pathogens 2022, 11, 671
weight of the sampled piglet”, and “parity of the corresponding dam” or “life‐borne pig‐
lets in the corresponding litter”, “dead‐borne piglets in the corresponding litter” and
“bodyweight of the sampled pig”. These examinations revealed that the viral load of
PCV3 in the tissue pools increased with the increasing age of the sampled piglets (rs:
−0.413; p = 0.045). In the generalized mixed model, this observation turned to a tendency
5 of 10
(p = 0.058). No further significant correlation was observed. The Cq values for the tissue
pools under consideration of the age of the sampled piglet are shown in Figure 1.
Figure1.1.Cq
Cqvalues
valuesof
oftissue
tissuepools
pools(PCV2
(PCV2and
andPCV3
PCV3PCR)
PCR)ininregard
regardto
tothe
theage
age(1–7
(1–7days
daysof
ofage)
age)of
of
Figure
the
sampled
piglets
(circle
and
asterisk
indicate
outliers).
the sampled piglets (circle and asterisk indicate outliers).
2.4. Presence of PCV2 and PCV3 DNA in OFS of Finisher Pigs
2.4. Presence of PCV2 and PCV3 DNA in OFS of Finisher Pigs
In grow–finish pigs, pen-based OFS were used to check for PCV2 and PCV3 DNA. All
In grow–finish pigs, pen‐based OFS were used to check for PCV2 and PCV3 DNA.
but one farm (15/16) were positive for PCV2 and PCV3 based on the PCR results of the OFS.
All but one farm (15/16) were positive for PCV2 and PCV3 based on the PCR results of the
The examination of the OFS independently from the time of sampling revealed that 72.6%
OFS. The examination of the OFS independently from the time of sampling revealed that
(95% CI: 69.3–76.3%) and 67.2% (95% CI: 62.9–71.1%) were PCV2 or PCV3 DNA-positive,
Pathogens 2022, 11, x FOR PEER REVIEW
6 of 11
72.6%
(95% CI:
and 67.2%
(95% CI: 62.9–71.1%)
werepositive
PCV2 or
DNA‐
respectively.
In 69.3–76.3%)
total, 51.1% (95%
CI: 46.4–55.3%)
of the OFS were
forPCV3
both viruses.
positive,
respectively.
total,
51.1% (95%
CI: 46.4–55.3%)
of the
OFS
wereof
positive
The qualitative
results In
of the
molecular
examinations
of the OFS
with
respect
the timefor
of
both
viruses.
The
qualitative
results
of
the
molecular
examinations
of
the
OFS
with
respect
sampling are shown in Table 5. An overview on the Cq values of all positive OFS are
given
of
time2.of sampling are
shown in Table 5. An overview on
the Cq values of all positive
(99/126)
(71/126)
(58/126)
inthe
Figure
OFS are given in Figure 2.
Table 5. Number and percentage of PCV2 or PCV3 DNA‐positive OFS or both of all farms at differ‐
ent times of sampling.
Week of
Life
6
12
16
20
PCV2 DNA‐
Positive OFS
% (n)
65.6%
(82/125)
67.2%
(90/134)
79.2%
(103/130)
78.6%
95% Confidence
Interval
%
56.8–73.6
59.0–74.6
71.7–86.2
71.4–85.7
PCV3 DNA‐
Positive OFS
% (n)
65.6%
(82/125)
75.4%
(101/134)
70.8%
(92/130)
56.3%
95% Confidence
Interval
%
56.8–73.6
68.7–82.8
63.1–78.5
47.6–64.3
PCV2 + PCV3 DNA‐
Positive OFS
% (n)
44.8%
(56/125)
56.0%
(75/134)
56.9%
(74/130)
46.0%
95% Confidence
Interval
%
36.0–53.6
47.8–63.4
48.5–65.4
37.3–54.8
Figure
2. Cq
values
single
PCV2
PCV3
DNA‐positive
OFS
growing
and
fattening
pigs
Figure
2. Cq
values
of of
single
PCV2
or or
PCV3
DNA-positive
OFS
of of
growing
and
fattening
pigs
at at
different
times
sampling.Each
Eachdot
dot represents
represents the
the Cq
lines
rep‐
different
times
ofof
sampling.
Cq value
value of
ofaasingle
singleOFS.
OFS.Horizontal
Horizontal
lines
resent the
represent
themedian
medianCq
Cqvalue
valueat
atthe
theparticular
particulartime
timeof
ofsampling
sampling (*
(* mean
mean Cq
Cq value).
value).
3. Discussion
The present study examined the occurrence of PCV2 and PCV3 DNA in suckling pig‐
lets crushed by their mothers within their first week of life, and includes the results of the
PCV detection in OFS in downstream production steps. The rationale for this kind of sam‐
pling was to bring our study in line with the “the three Rs” desired for scientific working
Pathogens 2022, 11, 671
6 of 10
Table 5. Number and percentage of PCV2 or PCV3 DNA-positive OFS or both of all farms at different
times of sampling.
Week of
Life
PCV2 DNAPositive OFS
% (n)
95% Confidence
Interval
%
PCV3 DNAPositive OFS
% (n)
95% Confidence
Interval
%
PCV2 + PCV3
DNA-Positive OFS
% (n)
95% Confidence
Interval
%
6
65.6%
(82/125)
56.8–73.6
65.6%
(82/125)
56.8–73.6
44.8%
(56/125)
36.0–53.6
12
67.2%
(90/134)
59.0–74.6
75.4%
(101/134)
68.7–82.8
56.0%
(75/134)
47.8–63.4
16
79.2%
(103/130)
71.7–86.2
70.8%
(92/130)
63.1–78.5
56.9%
(74/130)
48.5–65.4
20
78.6%
(99/126)
71.4–85.7
56.3%
(71/126)
47.6–64.3
46.0%
(58/126)
37.3–54.8
3. Discussion
The present study examined the occurrence of PCV2 and PCV3 DNA in suckling
piglets crushed by their mothers within their first week of life, and includes the results
of the PCV detection in OFS in downstream production steps. The rationale for this kind
of sampling was to bring our study in line with the “the three Rs” desired for scientific
working with animals, as published in Directive 2010/63/EU of the European Parliament
and of the Council of 22 September 2010 on the protection of animals used for scientific
purposes. Although this type of sampling has its limitations, it enabled us to gain relevant
and interesting data. However, the number of PCV2 or PCV3 DNA-positive piglets might
be overrepresented, as it cannot be excluded that the detected pathogens impacted the
health of the piglets, making them more prone to being crushed. While this may be true
for individual piglets, it is not necessarily applicable to an entire farm. However, the
high number of farms with PCV2-(15/16) and PCV3-(9/16) positive litters indicates an
overall high prevalence of PCV2 or PCV3 in breeding farms. While high prevalence rates
of PCV2-positive suckling piglets have been reported before [5,6], this is one of the first
reports on the occurrence of PCV3 in suckling piglets. Concerning PCV2, our findings are
in contrast to our own results [8] and results from colleagues from the Netherlands [9].
However, this difference can be best explained by the differences in sample type among
the studies. Whereas pre-suckle sera were examined in the previously mentioned studies,
we used tissue samples, as viremia might not necessarily be present in PCV2-infected
piglets [10]. Furthermore, pre-suckle sera are a good measure to evaluate the number of
viremic piglets at the time of birth, but the time span of one week used in the present
study also includes the possibility of infection after birth. This topic was discussed in
detail by Dvorak et al. [5], who showed that piglets can be readily infected with PCV2 in
utero and that they are under the constant challenge of PCV2 after birth through contact
with infected sows and a contaminated farrowing environment. Moreover, even colostrum
can be PCV2-positive [20,21] and may possibly lead to early PCV2 infections in suckling
piglets [20]. The presence of PCV3 DNA in the colostrum samples of sows was also readily
reported by Kedkovid et al. [19]. In the aforementioned study, high PCV3 loads in colostrum
samples were associated with high viral loads in the corresponding sows. As we did not
collect blood samples from the sows, we cannot refer to this observation. Interestingly, the
number of PCV3 DNA-positive piglets was significantly lower than PCV2 DNA-positive
ones. While the high PCV2 DNA detection rate confirms the ubiquitous nature of PCV2
and the results from other colleagues [5,6], PCV3 seems not to be ubiquitous in that age
group. Referring to this, an interesting aspect of our study results is the observation of
tendentially increasing viral loads in correlation with the progressed age of the sampled
piglets. Although our study has a cross-sectional character, these findings indicate that early
infections might be of relevance for the spread of PCV3 within the following production
steps as the increasing viral loads give a hint to viral replication. On the other hand,
co-infections with both viruses did not lead to higher viral loads in the tissue samples of
the piglets, which indicates that the infection of a pig with both viruses does not enhance
Pathogens 2022, 11, 671
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replication at that age. Overall, the viral loads in the tissue samples appeared low range,
indicating subclinical disease rather than PCV-associated reproductive failure [1,22].
In grow to finish pigs, the prevalence for both PCV2 and PCV3 was high, which is in
line with several other field studies [23–25]. Interestingly, in all but one farm, PCV2 DNA
and PCV3 DNA were present in the grow–finish phase. Furthermore, a high proportion of
the OFS was positive for both PCV2 DNA and PCV3 DNA.
In our study, we identified PCV2 sow vaccination as a protective factor concerning
the number of PCV2 DNA-positive piglets up to one week of age, whereas the PRRSV
vaccination of sows seems to have a protective impact concerning the number of PCV3
DNA-positive piglets. Whereas the protection against PCV2 after sow vaccination against
the latter is in line with the results of Oliver-Ferrando et al. [13], who were able to show
positive effects concerning the viability and reproductive performance of piglets and sows,
respectively, the lack of any effect on the prevalence of PCV3 DNA-positive piglets indicates
no cross-protection against PCV3 after sow vaccination against PCV2. This is in line with
previous observations in German fattening farms, where no significant correlation between
PCV3 detection and the PCV2 vaccination status of the herds was observed [23]. Furthermore, it has been demonstrated that the PCV3 ORF2, coding for the immunodominant
capsid protein, shows no significant similarity with the corresponding ORF2 of most other
circoviruses, including PCV1 and PCV2 [14,26,27]. Interestingly, sow vaccination against
PRRSV was significantly associated with a lower number of PCV3 DNA-positive piglets.
These results indicate a possible role of PRRSV as a co-factor in case of early PCV3 infections
in piglets and might be correlated with the immunosuppressive nature of PRRSV [28],
which is antagonized by the specific vaccination measure. However, although this observation was significant, there is a need for further elucidation under controlled or more
standardized conditions.
4. Material and Methods
4.1. Farms and Animals
The study was carried out on 16 farms between July 2018 and August 2019. The
participation was voluntarily. The study took place in several federal states of Germany
and Austria. The farms were either conventionally farrow-finish farms (14/16) or farrowto-wean farms directly connected to a fattening farm (2/16). Farm details were recorded
by a questionnaire at the time of sample collection and are available in the supplementary
materials (Table S1). All farms fulfilled the legal requirements concerning the housing
conditions of the corresponding countries.
4.2. Collected Materials
4.2.1. Tissues
Participating farmers were asked to collect piglets that were crushed by their dams
within the first week of life. In total, 185 crushed piglets from 134 litters were obtained.
The piglets were stored frozen at −18 ◦ C on the farms until collection by the staff of the
Clinic for Swine of the LMU Munich when OFS and farm-specific data were also collected.
On arrival at the Institute for Veterinary Pathology of the LMU Munich, the piglets were
defrosted and tissue samples were collected and processed. The collected tissues included
inguinal lymph node, spleen, thymus, lung and myocardium. The samples were assigned
to two aliquots (one diagnostic and one back-up sample).
4.2.2. Oral Fluid Samples (OFS)
Oral fluid samples were collected in a cross-sectional manner from pigs at the age
of 6, 12, 16 and 20 weeks at each farm. One cotton rope (IDEXX, Westbrook, ME, USA)
was used to sample 20 pigs. In total, we collected OFS from approximately 200 pigs
per farm and age group. If more than 20 pigs were housed in one pen, two ropes were
placed. In total, 515 OFS were collected, resulting in a total sampled population of maximal
10,300 individuals from 16 farms. The number of collected OFS per farm is available
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from Table 1. OFS collection took place as described by Prickett et al. [29]. The collection
period lasted 25 to 30 min; afterwards, the lower, wet part of the rope was inserted into
the supplied plastic bag and squeezed manually to release the oral fluid. The sampled
OFs were decanted into supplied 5 mL centrifuge tubes and were centrifuged at 1560 g for
10 min to separate feed particles and other contaminants from the liquid. They were stored
at −20 ◦ C until further analysis.
4.3. Molecular Biological Examinations
All OFS and tissue samples were examined for PCV2- and PCV3-specific DNA. For
DNA isolation, the DNA Mini Kit (Qiagen) was used according to the manufacturer’s instructions. For virus detection, we used a quantitative duplex PCR (Virotype® PCV2/PCV3,
INDICAL BIOSCIENCE GmbH, Leipzig, Germany).
4.4. Statistics
Statistical calculations were performed with the software IBM SPSS Statistics version
28.0.1.0 for Microsoft® Windows. The prevalence was specified as absolute and relative
frequencies in percent with the 95% confidence interval. The significance level was 0.05.
Dichotomous variables were analyzed for possible associations with the PCR outcome
(positive/negative) by a chi2 test. In case more than one independent variable was associated with the dependent variable, a binary logistic regression was also conducted. An
overview of the factors and distribution of the examined population used is available as
supplementary material (Table S2).
Independent factors that were significantly associated within the chi2 test were also
used to evaluate whether these factors not only had an influence on the qualitative outcome
concerning the PCV2 and PCV3 PCR, but also on the quantitative outcome (Cq values of
PCV2 and PCV3 PCR in tissue pools). Therefore, we conducted a multifactorial generalized
mixed linear model (gamma regression as target distribution) and added the independent
factor “both viruses in the same tissue pool”. For these examinations, only PCR-positive
tissue samples were included.
To assess whether the quantitative PCR outcome was potentially associated with
any external factors, we conducted a bivariate non-parametric correlation according to
Spearman, including “age of the sampled piglet”, “bodyweight of the sampled piglet”
and “parity of the corresponding mother”. The results were verified in a multifactorial
analysis, performing the generalized mixed linear model (gamma regression as target
distribution) including the individual farms as random factors. Furthermore, we checked
whether the Cq values had any impact on the dependent variables “life-borne piglets in the
corresponding litter”, “dead-borne piglets in the corresponding litter” and “bodyweight of
the sampled pig”, also by bivariate non-parametric correlations. For these examinations,
only PCR-positive tissue samples were included.
5. Conclusions
The present data confirm the ubiquitous nature of PCV2 in suckling piglets and
grow–finish pigs. In contrast, for PCV3, this observation was only true for grow–finish
pigs, whereas the PCV3 prevalence was low in suckling piglets. Co-infections with PCV2
and PCV3 in the same animals were of low prevalence and had no significant effect on
the viral loads or other parameters checked in this study. Sow vaccination against PCV2
was identified as a protective factor concerning PCV2, but not for PCV3, indicating no
cross-protection of PCV2 vaccination against PCV3. On the other hand, the vaccination
of sows against PRRSV was a protective factor concerning the PCV3 detection rate in
suckling piglets, indicating that early PCV3 infections might be modulated by this specific
pathogen interaction.
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Supplementary Materials: The following supporting information can be downloaded at: https:
//www.mdpi.com/article/10.3390/pathogens11060671/s1, Table S1: Overview of the single farm
specifications (ff: farrow to finish farm; Ery: Erysipelothrix rhusiopathiae; PPV: porcine parvovirus; IAV:
influenza A virus), Table S2: Overview of independent factors used for the statistical analysis. Targets
were the PCV2 and PCV3 PCR results (nominal/metric).
Author Contributions: Conceptualization, M.E., V.C., R.K. and T.O.; formal analysis, M.E., S.R. and
R.F.; funding acquisition, M.E.; investigation, R.M., P.D., L.E., V.C. and R.F.; project administration,
M.E. and W.S.; resources, L.E., V.C. and R.F.; supervision, M.E., W.S. and M.R.; validation, S.R.;
writing—original draft, M.E.; writing—review and editing, R.M., P.D., L.E., S.R., V.C., R.K., T.O., W.S.,
M.R. and R.F. All authors have read and agreed to the published version of the manuscript.
Funding: This study was funded by Ceva Tiergesundheit, Duesseldorf, Germany and Ceva Santé
Animale, Libourne, France. Additionally, T.O. was funded by Biotechnology and Biological Sciences
Research Council (BBSRC) (award numbers BBS/E/D/20002173 and BBS/E/D/20002174) awarded
to the Roslin Institute, University of Edinburgh.
Institutional Review Board Statement: The present study was requested and approved according to
national animal welfare acts by the appropriate authorities. Bavaria, Germany: Upper Bavarian Government, reference number ROB-55.2-2532.Vet_03-18-43, June 2018; Lower Franconia Government,
reference number RUF-55.2.2-2532-2-742-4, Nov. 2018. Baden Wuerttemberg, Germany: Regional
Council Tuebingen, reference number 35//9185.82-4, July 2018; Regional Council Stuttgart, reference
number 35-9185.82/0352, June 2018. North Rhine-Westphalia, Germany: North Rhine-Westphalia
State Office for Nature, Environment and Consumer Protection, reference number 81-02.05.40.18.056,
July 2018. Lower Saxony, Germany: Lower Saxony State Office for Consumer Protection and Food
Safety, reference number 33.19-42502-05-18A303, May 2018. Schleswig-Holstein, Germany: Ministry
for Energy Transition, Agriculture, Environment, Nature and Digitalization, reference number V24423216/2018, May 2018. Upper Austria, Austria: Federal Ministry Education, Science and Research,
reference number GZ 68.205/0098-V/3b/2019, May 2019.
Informed Consent Statement: Not applicable.
Data Availability Statement: The data presented in this study are available on reasonable request
from the corresponding author.
Acknowledgments: We would like to thank all persons involved in the study, especially the farmers
and the local veterinarians for their support.
Conflicts of Interest: The authors confirm that they have no conflict of interest. R.K. and V.C. are the
employees of Ceva Santé Animale and Ceva Tiergesundheit, respectively. R.K. and V.C. were not
involved in the statistical analysis.
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