by the American Association for Laboratory Animal Science
Vol 60, No 1
Porcine respiratory disease complex (PRDC) is an economically
significant problem characterized by slow growth, poor food uti-
lization, lethargy, anorexia, fever, cough, and dyspnea in pigs 16
to 22 wk of age.14,38 PRDC is associated with complex sequential
or concurrent infections with multiple viral or bacterial respira-
tory pathogens.6,8,17,29 Field investigations and case-trend analyses
demonstrate that porcine circovirus type 2 (PCV2) plays a role in
PCV2 belongs to the family Circoviridae, which contains the
smallest nonenveloped, single-stranded, circular DNA viruses.21,39
In the late 1990s, a pathogenic circovirus designated PCV2 was
isolated, which differed from the nonpathogenic PCV1.2,21 PCV2
is considered ubiquitous and can be detected in both diseased
and clinically healthy pigs.1 Infection induces various degrees of
lymphoid depletion and immune suppression, demonstrated by
experimentally infecting pigs with PCV2 infectious DNA clones.10
However, the factors that contribute to the pathogenicity of PCV2
remain unknown.24 Generally, infection with PCV2 alone is lim-
ited in its ability to induce the full spectrum of symptoms asso-
ciated with PRDC; the role of PCV2 in PRDC always involves
interaction or synergism with other respiratory pathogens.4,17
Swine influenza virus (SIV) is a common pathogen associated
with PRDC.6,8 SIV is an enveloped, negative-sense, segmented
RNA virus belonging to the family Orthomyxoviridae.36 SIV infects
the epithelium of the respiratory tract of pigs, causing an acute
infection with clinical signs of cough, fever, lethargy, and anorexia
beginning 1 to 2 d after experimental infection and lasting for 3
to 4 d.14,44 High morbidity and low mortality are quite common in
uncomplicated disease, but mortality usually is high when oth-
er infectious agents are present along with SIV.36 Together with
PCV2, SIV frequently is found in pigs with clinical signs of PRDC.
At one farm, mortality reached as high as 10% in pigs coinfected
with PCV2 and SIV, and 5% of the coinfected pigs failed to reach
market weight.15 In a cross-sectional study, SIV infection was 11
times more likely to occur in PCV2-positive pigs compared with
PCV2-negative pigs.8 Field studies on pigs with PRDC conducted
in different years showed a 1.9% to 13% rate of coinfection with
PCV2 and SIV.6,9,15,17,28,29 Clinical evidence suggests that SIV acts
synergistically with PCV2 to cause PRDC. However, the patho-
genesis of PCV2–SIV coinfection is unknown.
In this study, our goal was to establish a challenge model for
PCV2 and PCV2–SIV and to determine whether SIV influences
PCV2 replication and increases the severity of PRDC. Throughout
the study, microscopic lesions attributable to PCV2 and PCV2 vi-
ral load in serum, nasal swab, lung, and lymph node did not dif-
fer between PCV2- and PCV2–SIV-inoculated pigs. On the basis
of these findings, we conclude that SIV H1N1 did not influence
PCV2 replication in dually infected animals.
Materials and Methods
Experimental design. Cesarean-derived colostrum-deprived
Large White pigs were obtained from Struve Labs (Manning, IA)
and were randomly assigned to 3 groups of 8 pigs each after they
were delivered to the livestock infectious disease isolation facility
at Purdue University (West Lafayette, IN), which is a Biosafety
Level 2 animal housing facility with negative-pressure HEPA-
Infection of Cesarean-Derived Colostrum-Deprived
Pigs with Porcine Circovirus Type 2 and
Swine Influenza Virus
Huiling Wei, Stephen D Lenz, William G Van Alstine, Gregory W Stevenson,†
Ingeborg M Langohr,‡ and Roman M Pogranichniy*
Porcine circovirus type 2 (PCV2) and swine influenza virus (SIV) are important pathogens for porcine respiratory disease complex,
which is economically significant worldwide. The pathogenesis of PCV2–SIV coinfection is unknown. In this study, we focused
on establishing a challenge model for PCV2 to determine whether SIV influences PCV2 replication and increases the severity of
PCV2-associated disease. Cesarean-derived colostrum-deprived pigs were inoculated intratracheally with cell culture medium only
(negative control group), PCV2 only, or PCV2 followed 1 wk later with SIV H1N1. Two pigs from each group were necropsied at
12, 21, 28, and 35 d after inoculation. Coinfection with SIV did not increase the number of PCV2 genomic copies in serum or target
tissues or the severity of microscopic lesions associated with PCV2 in lung or lymph node. The antibody titer to PCV2 did not
differ significantly between PCV2–SIV- and PCV2-infected groups. In conclusion, SIV H1N1 did not influence PCV2 replication
in dually infected pigs in this study.
Abbreviations: PCV2, porcine circovirus type 2; PRDC, Porcine respiratory disease complex; SIV, Swine influenza virus.
Received: 28 Aug 2009. Revision requested: 28 Sep 2009. Accepted: 09 Nov 2009.
Department of Comparative Pathobiology, Purdue University, West Lafayette, Indiana.
*Corresponding author. Email: firstname.lastname@example.org
Current affiliation: †Department of Veterinary Diagnostic and Production Medicine, Iowa
State University, Ames, Iowa; ‡Diagnostic Center for Population and Animal Health,
Michigan State University, Lansing, Michigan.
Vol 60, No 1
infecting PK15 cells with PCV2 and fixing infected cells to the
bottoms of the wells by using 80% aqueous acetone. Antigen–an-
tibody reaction was visualized by staining cells with fluorescein-
conjugated goat-antiswine PCV2 antibody (Kirkegaard and Perry
Laboratories, Gaithersburg, MD). Antibody titers for individual
samples were determined as the reciprocal of the highest dilution
that showed specific fluorescence. The presence of SIV antibody
in serum samples was tested by hemagglutination inhibition as-
say as described previously.45
Virus isolation and real-time PCR assay. Nasal swabs were col-
lected on study days 7, 10, 12, 15, 17, 19, and 21. Inguinal, sub-
lumbar, mesenteric, tracheobronchial, and submandibular lymph
nodes and lung collected at necropsy (days 12, 21, 28, and 35; 2
pigs from each group per day) were frozen at –80 °C until pro-
cessing. Viral DNA and RNA were extracted (QiaAmp DNA Mini
Kit and QiaAmp Viral RNA Mini Kit, respectively; Qiagen, Santa
Clarita, CA) from serum, nasal swabs, and tissue homogenate
from pooled lung or lymph node samples. Real-time PCR for
PCV226 and SIV H1N132 were used to quantify the viral genome
copies in each sample. Freshly prepared pooled lung or lymph
node tissue homogenate was used for virus isolation for both
PCV2 and SIV. For PCV2, tissue homogenate was inoculated into
a PK15 cell suspension and cultured for 3 d; infected cells were
treated with 300 mM D-glucosamine, incubated 24 h longer,40 and
used in immunofluorescent assays. SIV was isolated from lung
homogenate as previously described.45
Histopathology. On study days 12, 21, 28, and 35 d, 2 pigs from
each group were weighed, euthanized, and necropsied and a
mean group body weight determined by using the weights of the
2 representative pigs euthanized.
Lung and lymph node samples from all pigs were fixed in 10%
neutral buffered formalin, processed, and embedded in paraffin,
and sections were stained with hematoxylin and eosin (Animal
Disease Diagnostic Laboratory, West Lafayette, IN). Lung and
lymph node lesions attributable to PCV2 infection were evaluated
by a board-certified veterinary pathologist, and scored by using a
semiquantitative scoring system. Airway lesions in lung epithelial
(necrosis, hypertrophy, hyperplasia, intraepithelial leukocytes,
periglandular inflammation) were scored as: 0, absent; 1, mild
(0% to 10% of the airways affected); 2, moderate (11% to 30% of
airways affected); 3, marked (31% to 50% of airways affected);
and 4, severe (more than 50% of the airways affected). Pulmonary
inflammation was categorized as suppurative, lymphoplasma-
cytic or histiocytic; each category of inflammation was scored as:
0, absent; 1, mild; 2, moderate; and 3, marked. Individual airway
lesion and inflammation scores were summed for each animal to
give an overall score for the pulmonary alterations for each ani-
mal. Lymph node lesions (lymphoid depletion, lymphohistiocytic
or granulomatous lymphadenitis) were scored as: 0, absent; 1,
mild; 2, moderate; and 3, marked. Scores for lymphoid depletion
and inflammation were summed for each animal to give an over-
all score for lymph node alterations for each animal.
In situ hybridization for PCV2. PCV2 nucleic acid in lung and
lymph nodes was detected by in situ hybridization as previously
described13,31 with some modifications. After deparaffinization
and rehydration, tissue sections on slides were prehybridized at
42 °C for 1 h. Hybridization was performed overnight at 42 °C
with digoxigenin-labeled oligoprobes specific for the PCV2 genes
encoding the replicase and capsid proteins.18 Antidigoxigenin
antibody conjugated with alkaline phosphatase (dilution 1:500)
filtered ventilation. The experimental protocol was approved
by the Purdue Animal Care and Use Committee. All the rooms
and equipment in the isolation facility were disinfected twice
(1% Virkon S, Pharmacal Research Laboratories, Waterbury, CT)
followed by thorough washing before the delivery of the pigs.
Each group of pigs was housed together on raised wire decks
in a single, isolated room equipped with a nipple drinker and a
self-feeder. Feed that was neither autoclaved nor irradiated was
delivered by Struve Labs. The pigs were kept at 75 to 79 °F on a
12:12-h light:dark cycle. Before the study, the pigs were negative
serologically (by ELISA) and by quantitative real-time PCR to
most PRDC-related pathogens: PCV2, SIV, porcine reproductive
and respiratory virus, and M. hyopneumoniae. At the end of the
experiment, all pigs remained negative for porcine reproductive
and respiratory virus and M. hyopneumoniae. The respective ex-
perimentally inoculated groups were positive for PCV2 and SIV
(by serologic assay), and PCV2-inoculated groups were positive
by real-time PCR for the presence of circovirus DNA in serum;
negative control animals remained negative for SIV and PCV2
throughout the study.
One week after delivery (study day 0), pigs (age, 7 wk) were
sedated by intramuscular injection (0.2 mL/12 lb. body weight)
of a mixture of telazol, ketamine, and xylazine (final concentra-
tion of each drug, 50 mg/mL). Sedated animals were placed in
sternal recumbency, the jaws were opened manually, the larynx
was sprayed lightly with topical anesthetic (Cetacine, Cetylite
Industries, Pennsauken, NJ), and a lighted oral speculum was
placed in the mouth to facilitate insertion of a semirigid catheter
into the proximal trachea, through which the virus suspension
was to be deposited into the trachea. Pigs in groups 1 (5 female,
3 male) and 2 (3 female, 5 male) each were inoculated with 3 mL
PCV2 genotype 1 (strain ADDLPP 10069, 1.6 × 105 TCID50/mL),
whereas those in group 3 (2 female, 6 male) received 3 mL culture
medium only. The following week (study day 7), pigs (age, 8 wk)
again were anesthetized and inoculated with PCV2 as previously
(groups 1 and 2) or culture medium (group 3); group 1 animals
also received 3 mL SIV H1N1 (A/Swine/A07-7967/IN, 2 × 107
pfu/mL). Pigs were kept under strict Biosafety Level 2 condi-
tions to avoid cross-contamination among groups. In particular,
appropriate personal protective equipment (coveralls, respirator
[N95] masks, hairnets, latex gloves, and boots) and a designated
room-entry order (group 3 > group 2 > group 1) were used by all
personnel. People who had been in contact with other animals
during the previous 24 h were required to shower before entering
the pig isolators.
On the day of necropsy, pigs were euthanized by intraperito-
neal injection (1 mL/10 lb. body weight; Beuthanasia D Special,
Schering–Plough Animal Health, Union, NJ) and, after a surgical
plane of anesthesia was achieved (that is, lateral recumbency and
loss of palpebral, swallow, and gag reflexes), exsanguination by
severing the axillary artery.
Clinical evaluation. Clinical signs (behavior, dyspnea, and
cough) were monitored and scored (1, normal; 2, mild change; 3,
marked change) daily.
Serologic assays. Immunofluorescent and hemagglutination inhi-
bition assays. Blood for serum samples was collected from jugular
veins or at necropsy on study days 0, 7, 12, 21, 28, and 35. PCV2
antibody titer was assessed by immunofluorescent assay as pre-
viously described.31 Briefly 4-fold dilutions (starting at 1:20) of
serum samples were applied to 96-well titer plates prepared by
Swine model of coinfection with PCV2 and SIV
red-purple, slightly firm, and slightly depressed. On section, le-
sions typically were peribronchial, and bronchi contained clear to
slightly opaque, catarrhal exudates. Pigs in the PCV2 and nega-
tive-control groups lacked gross lung lesions. Tracheobronchial
lymph nodes were enlarged in pigs in both the SIV–PCV2 and
Microscopic lesions attributable to PCV2 infection consisted of
lymphoid depletion, lymphohistiocytic or granulomatous lymph-
adenitis, and interstitial pneumonia. According to semiquanti-
tative scoring, the microscopic lesions associated with PCV2 in
lymphoid tissues (Figure 5) and lungs (Figure 6) were not signifi-
cantly different between PCV2–SIV and PCV2 groups (data not
shown). The intensity of PCV2 signal by in situ hybridization of
lung and lymph node also did not differ significantly between the
2 groups (data not shown).
and the substrates nitroblue tetrazolium and 5-bromo-4-chloro-
3-indolyl phosphate (Roche Diagnostics, Indianapolis, IN) were
applied to visualize the presence of PCV2 nucleic acid.
Statistical analysis. Differences in body weight and PCV2 vi-
ral load in nasal swabs, lung, and lymph nodes were evaluat-
ed by comparing the means with one-way ANOVA (Prism 4.0,
GraphPad Software, La Jolla, CA). Any statistical values P < 0.5
was considered statistically significant in this study. Clinical and
pathologic scores and PCV2 nucleic acid signals by in situ hybrid-
ization were analyzed by using the Wilcoxon rank-sum test (SAS
9.1.3, SAS Institute, Cary, NC).
Clinical evaluation. The negative control group (group 3) re-
mained healthy throughout the study. Pigs inoculated with only
PCV2 (group 2) showed mild and transient respiratory disease
without coughing. Pigs dually infected with PCV2 and SIV
(group 1) exhibited mild to moderate respiratory signs of PRDC
characterized by increased respiratory rate, lethargy, and occa-
sional coughing. Clinical signs for behavior and coughing did
not differ significantly between PCV2- and PCV2–SIV-groups
(data not shown), but respiratory scores were significantly (P <
0.05) higher for PCV2–SIV-infected pigs than those in the PCV2
group from days 9 to 23 (Figure 1). Respiratory disease lasted 4
times longer in the PCV2–SIV group than in PCV2 group. The
group mean body weight of the 2 pigs at necropsy did not differ
between groups (Figure 2).
Serologic assays. Immunofluorescence and hemagglutination
inhibition assays. All pigs were serologically negative for PCV2
and SIV before inoculation. On day 12, 6 of the 8 pigs in the PCV2
group and 3 of the 8 pigs in the PCV2–SIV group had seroconver-
ted to PCV2. By day 21, 7 of the 8 PCV2–SIV-infected pigs and all
8 pigs in the PCV2 group had developed detectable PCV2 anti-
body responses. The mean antibody titer to PCV2 did not differ
between the 2 groups throughout the study (Figure 3). Antibodies
to SIV were first detected in all pigs in PCV2–SIV group on day
21 after SIV infection.
Virus isolation and PCR. Negative-control pigs (group 3) re-
mained negative for both PCV2 and SIV throughout the study.
On day 7, all 8 pigs in the PCV2 group and 4 of the 8 compris-
ing the PCV2–SIV group were viremic for PCV2. All pigs in both
groups were viremic by day 12, and the viremia of the pigs in
both groups persisted for the entire 35 d of the study. PCV2 shed-
ding was detected in all nasal swabs (obtained on days 7, 10, 12,
15, 17, 19, and 21) from all the pigs in both the PCV2–SIV and
PCV2 groups. The PCV2–SIV and PCV2 groups did not differ sig-
nificantly in the mean number of PCV2 genomic copies in serum,
pooled lung, pooled lymph node, and nasal swabs (Figure 4).
Real-time PCR detected SIV in nasal swabs collected from
PCV2–SIV-infected pigs on days 10 and 12 (3 and 5 d after SIV
inoculation) and in pooled lung homogenates on day 12. In addi-
tion, SIV was isolated from the lungs of PCV2–SIV-infected pigs
euthanized on day 12. On day 35 (study end), PCV2 was isolated
from all lymph node samples, 6 of 8 lung samples from PCV2–
SIV-infected pigs, and from 5 of 8 lungs from pigs in the PCV2
Histopathology. In the PCV2–SIV group, gross lung lesions
were lobular in distribution; were sharply demarcated from adja-
cent, nonpneumonic lung; and primarily affected the dependent
regions of the apical and cardiac lobes. Affected tissue was dark
Figure 1. Mean daily clinical respiratory scores from day 1 to study end.
*, P < 0.05; between values for negative-control and SIV–PCV2-infected
Figure 2. Mean body weight of the 2 pigs from each group euthanized
on days 12, 21, 28, and 35.
Figure 3. Mean serum PCV2 antibody titer measured by immunofluo-
Vol 60, No 1
Figure 4. Group mean PCV2 viral load in serum, lung, lymph node, and nasal swabs.
Figure 5. Lymph node, day 28. (A) Representative section from the negative-control group shows a normal lymphoid follicle. Representative sections
from (B) SIV–PCV2-infected and (C) PCV2-infected pigs show similar lesions, with mild lymphoid depletion and few histiocytes and multinucleated
giant cells infiltrating lymph node follicles. Hematoxylin and eosin stain; bar, 50 μm.
Figure 6. Lung, day 35. Representative sections from (A) SIV–PCV2-infected and (B) PCV2-infected pigs. Both sections show moderate peribronchiolar
lymphoplasmacytic inflammation and mild mononuclear leukocyte infiltrates in the alveolar septa, consistent with interstitial pneumonia. Hematoxy-
lin and eosin stain; bar, 100 μm.
Swine model of coinfection with PCV2 and SIV
however, given the many interacting variables of infection includ-
ing strains of viruses, age at infection, relative timing of infection,
and immune reactivity of individual pigs, additional studies are
needed to model and study the disease.
In addition, to obtain deeper understanding of the pathogen-
esis of concurrent infection with PCV2 and SIV, comprehensive
epidemiologic studies should be done by measuring and moni-
toring important parameters including pig age, the approximate
time of infection by PCV2 and other common respiratory viruses,
viral titers, vaccinations, and immune response—all of which
may influence the disease outcome. The rate and consequences
of PCV2–SIV coinfection differs from pig to pig and from herd to
herd, and these different parameters should be explored further
to understand PRDC disease development in the field and to help
develop models for future study.
The authors thank the staffs of the Virology and Serology Section
(Animal Disease Diagnostic Laboratory) and Veterinary Lab Animal
Care (Purdue University, West Lafayette, IN) for their help during this
study. The study was supported by School of Veterinary Medicine
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Field case studies have shown that coinfection of pigs with
SIV and PCV2 is associated with severe PRDC.15,17 In the present
study, infection with PCV2 only caused mild respiratory signs,
whereas PCV2–SIV-infected pigs showed severe and prolonged
disease. This difference might reflect synergism between SIV and
PCV2. However, scoring of lesions in lung and lymph nodes and
the PCV2 viral load in lung, lymph node, and nasal swabs were
not significantly different between the PCV2- and PCV2–SIV-in-
fected groups. Therefore, the data of this study indicate that the
H1N1 strain of SIV does not enhance the replication of PCV2,
and there is limited synergism between the 2 viruses. There may
be multiple known or unknown factors complicating the clinical
manifestation of PCV2–SIV coinfections in the field.
The SIV strain in the conditions of this study is weakly patho-
genic. Due to insufficient replication, SIV may not have induced
sufficient cytokines (including such as IFNα, IL6, and TNFα) or
monocyte-attracting chemokines to potentiate PCV2 replication
in the lung.42,43 The low pathogenicity of the SIV strain used may
have limited pulmonary cell death and subsequent cellular regen-
eration, resulting in fewer actively replicating cells in lung than
typical during severely pathogenic swine influenza. This decrease
would be significant because PCV2 DNA synthesis requires cel-
lular enzymes that are expressed during the S-phase growth of
actively replicating cells.40 The lack of these enzymes to enhance
replication of PCV2 might account for the failure of SIV to poten-
tiate PCV2 replication in our model.
The timing of coinfection might also be important in the induc-
tion of PRDC.25 In PCV2 infection, viral load has been correlated
with disease severity.3,23,34 Perhaps with a longer interval between
inoculation with PCV2 and SIV the pigs might have developed a
PCV2 viral load high enough to interact synergistically with SIV.
In addition, the age of pigs when they are infected with PCV2
may influence disease development.31,41 A meta-analysis of ex-
perimental infections with PCV2 found that age younger than
3 wk at inoculation showed strong positive correlation with the
development of disease.41 Because of the terms of our contract
with the vendor, we could not obtain cesarean-derived colostrum-
deprived pigs younger than 5 wk; future studies likely should
involve pigs 3 wk of age and younger. Although disease severity
is inversely correlated to the magnitude of the PCV2 antibody
response,22,30 the cause of the weak immune response in severely
diseased pigs is unknown. All pigs in the present study devel-
oped strong humoral immunity to PCV2 (Figure 3) and lesions
typical of PCV2-associated disease, except wasting. Animal hus-
bandry also plays a role in the development of disease.24
The results of this study suggest that coinfections of SIV and
PCV2 in severe cases of PRDC are underestimated by the routine
diagnostic testing done in the field. Infection with PCV2 is ubiqui-
tous in both clinically healthy and sick pigs all over the world.29,33
Infection with SIV is also common. Serologic surveillance for SIV
reveals a prevalence ranging from 20% to 100% in pig populations
of different ages in different countries.5-7,11,16,19,20,27,35,37 Therefore, the
likelihood of the 2 viruses coinfecting the same pig population is
high. In contrast, the reported rate of concurrent SIV-PCV2 during
PRDC in the field is 1.9% to 13%.6,9,15,17,28,29 In the present study, SIV
was cleared rapidly but PCV2 was not. Perhaps similarly in field
cases of PRDC, the SIV infection often has cleared before routine
diagnostic testing is done on clinically ill animals. Coinfection
with PCV2 and SIV remains a likely common cause of PRDC;
Vol 60, No 1 Download full-text
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