Variation in Response to Infection in Experimental Challenges with Porcine Circovirus 2b
Individual variation in the magnitude and time of immune response was demonstrated in experimental challenges with PCV2b, report Theresa P. Bohnert and colleagues at the University of Nebraska in the Nebraska Swine Report 2011.Summary
Porcine Circovirus 2 is the
etiological agent of many associated
diseases that impact performance and
increase mortality. Vaccination is costly
and time-consuming.
In this study, 81
barrows of two commercial genetic lines
were infected with a PCV2b strain and
nine barrows receiving no inoculation
were controls. Blood was drawn weekly
and tested for levels of IgG, IgM, and
viraemia.
Infected pigs showed three
patterns of IgM response: early, late,
and limited response. Pigs showing no
response generally had faster growth
and lower viraemia, most likely due
to an inhibition of virus replication.
Early response individuals tended to
have lower viraemia and faster growth
compared to individuals with late
response. This important variation
in immune response has a potential
economic value and could be used in
management practices and breeding
programmes.
Future research will be
focused on dissection of genetic and
non-genetic factors explaining variation
in immune response and disease
susceptibility.
Introduction
Economic losses associated with
susceptibility to Porcine Circovirus
Associated Diseases
(PCVAD) continue
to have an impact on swine
industry.
Pigs affected by PCVAD
display characteristics of wasting,
diarrhea, interstitial pneumonia, dermatitis,
lymphoid depletion leading to
decreased immune responses, and susceptibility
to other pathogens. Porcine
Circovirus 2 (PCV2) is the causative
source of PCVAD, but additional factors
influence disease progression, of
which host genetics and secondary
immune stressors are highly important.
Secondary infection with swine
influenza, Mycoplasma hyopneumoniae
and Porcine Reproductive and
Respiratory Syndrome (PRRS) can
influence the severity and progression
of PCVAD.
Vaccines for PCVAD exist, but
they increase production costs, and
producers who utilize the vaccine may
still experience outbreaks. Even though
only 5 to 15 per cent of pigs infected with
PCV2 display clinical symptoms, the
whole herd must be immunized, which
is a costly practice. Recent research
has indicated that host genetics could
influence susceptibility to disease. For
example, several reports suggest that
Landrace pigs are more susceptible to
PCVAD than Pietrain and Duroc pigs.
These breed differences mean that
genetic variation within breeds likely
exists and that selection for resistance
may be possible. However, disease
resistance is difficult to improve using
traditional selection methods that
require regular disease challenges or
uniform and continued exposure to
the pathogen. Selection based on DNA
markers may be more effective. As long
as marker panels with known relationships
with immune response variables
are available, selection can be practiced
in any population without exposure to
the pathogen. Before DNA selection can
be implemented, comprehensive
disease phenotypes and DNA samples
need to be collected from pigs uniformly
exposed to PCV2b to determine
the relationships between DNA
markers and disease susceptibility.
This study investigated individual
response to experimental PCV2 infection
by profiling major indicators of
immune response in pigs from two
crossbred lines. This study represents
initial efforts to establish a large collection
of samples and PCVAD phenotypes
that will be used to identify
genes and DNA markers associated
with PCVAD resistance.
Materials and Methods
PCV2b isolate
Isolate UNLVBMS was recovered from a pig that had symptoms characteristic of PCV2b infection. Viral DNA was isolated using QIAamp DNA Minikit (Qiagen). Two pairs of primers were used for amplification of the entire PCV2b genome. DNA amplification was performed using GoTaq Flexi DNA Polymerase (Promega and PCR products were purified using ExoSAP-IT (USB Corporation). The viral DNA was sequenced from both directions using dye terminators and ABI PRISM 3100 Genetic Analyzer (Applied Biosystems).
Animals and facility
Animal use and experimental procedures were approved by the Institutional Animal Care and Use Committee of the University of Nebraska– Lincoln (UNL). The experiment included 91 crossbred barrows originated from 24 litters that were either Large White × Landrace (W×R, n=72) or a three-way cross with Duroc sires and W×R dams [D (W×R), n=19]. Pigs were born at the UNL Swine Farm and at approximately 35 days of age were transported to the animal science research facility where the disease challenge was conducted. Pigs at the farm are routinely tested for major pathogens and are known to be negative for PRRSV. The pigs were housed in one room and randomly allocated to 18 identical pens with a combination of slatted and solid surface flooring. The pens provided approximately 0.65 square metres of floor space per pig. All pigs were fed ad libitum using a standard balanced diet.
Experimental infection
The objective was to infect pigs
with virus after maternal antibodies
had waned and before natural infection
had occurred.
Blood was drawn
from pigs before infection and analyzed
for IgG (maternal antibodies)
and IgM (self-antibodies produced in
response to infection) to determine
when to initiate infection with PCV2b.
The sample-to-positive (S/P) ratios
of the maternal antibodies (IgG) in
all individuals selected for the experiment
were less than 0.3 at the time of
inoculation, the level specified by the
Ingezim ELISA protocol, which indicates
that protection from maternal
antibodies had waned. S/P ratios of
IgM were less than 0.4, indicating that
natural infection had
not occurred.
The virus inoculum contained the
titer of 104.0 50 per cent tissue culture infection
dose (TCID50)/mL in minimum
essential media with 50µ/mL gentamicin
and five per cent foetal calf serum. The
original experimental protocol was to
infect each pig with 2mL of inoculum
intramuscularly (IM) and 3mL
intranasally (IN). However, approximately
20 minutes after inoculation
of the first 20 pigs, anaphylactic shock
occurred and
eight of the pigs died.
The dose administered to the remaining
pigs was therefore altered. Most
of the pigs (n=58) received 1mL
IM and 3mL IN. The rest of the pigs
received a variation of this dose: 1mL
IM with 0.5 mL IN (n=4) or 2mL
IM with 0.5 mL IN (n=8). The pigs
used for these treatments were chosen
at random. Ten pigs were selected as
negative controls, assigned to separate
pens, and not inoculated.
Serology
Blood samples were collected before inoculation and at 7, 14, 21, and 28 days post innoculation (d.p.i.). Levels of PCV2 specific antibodies, IgG and IgM, were measured from serum using ELISA assays (Ingenasa). Samples were considered positive if the calculated S/P ratio was greater than 0.3 for IgG and 0.4 for IgM. Antibody data were normalized based on positive control values obtained for each plate.
Clinical evaluation and necropsy
Pigs were observed daily for clinical signs of infection, and weighed at 0, 7, 14, 21 and 28 d.p.i. Necropsy was performed at 28 d.p.i. Lung, spleen, and mesenteric and bronchial lymph nodes were collected for histology examination and gene expression analyses.
PCV2 quantification
Viral DNA was extracted from serum collected at 7, 14, 21, and 28 d.p.i. using QIAamp DNA Minikit (Qiagen). Estimates of the number of viral copies were obtained by quantitative real-time PCR using TaqMan Master Mix and ABI 7900 Real Time PCR System (Applied Biosystems). The area under the curve (AUC) was calculated to estimate total viral load throughout the 28-day experiment.
Statistical analysis
Least Square Means (LSM) were obtained using mixed-model procedures including the immune response pattern with crossbred lines as fixed effects and pen and litter as random effects. Analysis of the IgM antibody profiles during the challenge revealed that infected individuals are characterized by three patterns of immune response and could be separated in three groups:
- individuals that responded immediately to infection with the highest change in specific PCV2 IgM antibodies from 7 to 14 days post infection( d.p.i.) (n=33)
- individuals that responded late with the greatest change from day 14 to 21 (n = 40); and 3) individuals that did not respond to infection (n=7) (Figure 1).

Most pigs clearly fit into one of these groups, but in some cases pigs were placed in a group based on a subjective judgment. Correlations among traits were calculated from variances and covariances adjusted for line effects.

Results and Discussion
Genetic characterization of the PCV2b isolate
The PCV2b strain utilized in this experiment was isolated from a pig showing clinical symptoms of PCVAD. The viral DNA was isolated and sequenced having the highest genetic similarity with the PCV2b strain FMV-05-6507. This strain was first identified in 2005 in Quebec, Canada, and is known to induce clinical signs of postweaning multisystemic wasting syndrome (PMWS, a form of PCVAD) and increased mortality rate.
Anti-PCV2-IgM and –IgG antibodies
All candidate pigs for the PCV2b
challenge were produced by dams
that were vaccinated for PCVAD. The
offspring receive anti-PCV2 antibodies
via colostrum that provides
immunity against this pathogen. Its
effectiveness depends on the rate of
antibody decay that varies from five to
21 weeks. Seventy-five per cent of the
120 five-week-old candidate pigs for
experimental challenge had
levels of
maternal antibodies (IgG) below the
threshold (S/P ratio lower than 0.3)
that differentiate PCV2 negative from
positive pigs. Eighty-two per cent of the
W×R pigs (n=89) had IgG levels below
the threshold compared to 58 per cent of
the D (W×R) pigs (n=31). The levels
of IgG in the two crossbred groups did
not differ (P>0.10) before infection.
With the exception of day 7, pigs
in Group 3 had the lowest IgM values
during the challenge. Observed IgM
values calculated as sample to positive
ratios for each group at 0, 14, 21,
and 28 d.p.i. are presented in Figure
2. At day 0 and 7, IgM values were
similar, and at day 14, the controls
had the lowest value at 0.77, whereas
the Group 1 pigs had the highest at
1.38. The IgM values of the pigs from
Group 1 remained elevated at day 14,
and then the response began to wane
at day 21. The IgM values of Group
2 pigs reached the peak at day 21 and
declined thereafter. At day 21, the controls
and Group 3 pigs had the lowest
IgM values, 0.92 and 0.89, respectively.
At necropsy on day 28, the controls
and Group 3 pigs continued to have
the lowest IgM values, whereas Group
1 pigs had the highest and Group 2
had intermediate values.


Recent reports showed that rate
of maternal IgG decay varies considerably
between individuals and breeds,
which affects infection outcome and
disease progression. There were no
differences between groups in IgG level
at day 0 (Table 1). With the exception
of day 7, Group 3 had the lowest IgG
values during the challenge. Interestingly,
the IgG values of all individuals
from Group 3 were below the positive
threshold.
Differences in IgG levels
between groups occurred on day 14,
21, and 28 (P<0.05). IgG values for
infected and control pigs did not differ
at day 0, averaging approximately 0.77.
At day 7, the controls had the highest
values at 0.94, whereas the other three
groups remained steady around 0.75.
At 14 d.p.i, the controls were lowest
at 0.70 and the Group 2 pigs were the
highest at 1.83. The same trend continued
at 21 d.p.i.
The IgG response in
pigs in Group 3 was markedly different.
Values remained low until day 14,
increased to a value of 0.91 at day 14,
and then returned to baseline levels at
day 28. There were no significant differences
in IgM and IgG between the
two genetic lines, W×R and D (W×R), at any time point (Figures 3 and 4).


Amount of PCV2 DNA in serum samples
Following the experimental infection, quantitative PCR results for viral copy count showed that most of the barrows at day 7, 14, 21, and 28 d.p.i. tested positive for PCV2 DNA (Table 1). Differences in viral copy number between W×R and D (W×R) existed at day 7 (P<0.006) and day 14 (P<0.01). DNA copy number increased in both lines through day 21 and then declined (Figure 5).

Pigs in Group 3 had fewer viral
copies than pigs in the other groups.
At day 7, Group 1 had the highest
response at 4.56, whereas the response
in Group 3 was 3.85. At day 14, 21, and
28, Group 2 had the highest viral copy
count at 5.24, 6.17, and 4.7, respectively;
Group 3 remained the lowest
at 4.27, 4.67, and 3.96, respectively.
For viraemia AUC, the Group 3 pigs
were the lowest, with Group 1 being
intermediate and Group 2 being the
highest. viraemia of the control pigs
remained baseline
throughout the
study.
The amount of PCV2 administered
to pigs did not influence disease
progression, with the exception of
viraemia results
at day 7 (P<0.05).
However, there were only four pigs
in the group that differed from the
others,
and they received the lowest
viral dose of all 81 pigs infected.
Clinical evaluation
Only one death occurred during the four weeks of experimental challenge. This individual died during the last week of challenge, displayed wasting disease and had the highest viral load (AUC) in the first three weeks of the challenge. This individual displayed an early immune response and was inoculated with the lowest viral dose. The only difference in growth occurred between Groups 1 and 2 for average daily gain during the 28-day period (P<0.05). However, consistent with viraemia and IgM responses, pigs in Group 3, that had a limited immune response, had the greatest ADG.
Correlations
Correlations between average daily gain and viraemia results are summarized in Table 2. Moderately strong, negative correlation was estimated between average daily gain during the entire challenge and viral load (AUC) (r=-0.29).

Conclusion
The results of this preliminary
research provide strong evidence of
significant variation in immune response
in experimental challenges with
PCV2b. Analysis of the profiles of viral
load and antibody
response revealed
three groups of individuals that display
important variation in response to
infection. This variation is the source of
the differences in growth between pigs
during the challenge. The main difference
between individuals from Group
1 and 2 was in the time of response,
whereas the difference between these
two groups and Group 3 was in the
magnitude of response. The source of
limited immune response in individuals
from Group 3 is most likely a mechanism
that inhibited virus replication.
Individuals that responded immediately
to infection (Group 1) had greater ADG
than those that responded late (Group
2). Pigs in Group 3 had the greatest rate
of growth during the 28-day period
following infection, but the observed
differences were not statistically significant.
The objective of our future research
is to identify if the source of the
differences is genetic and to uncover
potential genes and genetic variants
responsible for PCVAD susceptibility.
This research will generate knowledge
that can be applied to other viral diseases
such as PRRSV. The long-term objective
is to generate a panel of genetic
markers that can be used in breeding
programs to improve genetic resistance
to PCVAD. The benefits of improving
PCVAD susceptibility using genetic
markers are obvious: lower production
costs associated with improved robustness
and a decrease in the number of
vaccinated pigs, fewer welfare issues,
and increased international competitiveness
of the US swine industry.
1Theresa P. Bohnert is a graduate student,
and Autumn M. McKnite is a research technician
in the UNL Animal Science Department;
Judith W. Galeota is research manager,
Timothy Moural, research technologist, and
Seth Harris, assistant professor in the UNL
School of Veterinary Medicine and Biological
Sciences; Rodger K. Johnson is a professor,
Thomas E. Burkey and Daniel C. Ciobanu are
assistant professors in the UNL Animal Science
Department.
Reference
Bohnert T.P., A.M. McKnite, J.G. Galeota, T.W. Moural, S.P. Harris, T.E. Burkey, R.K. Johnson and D.C. Ciobanu. 2011. Variation in response to infection in experimental challenges with porcine circovirus 2b. Nebraska Swine Report 2011, p31-35.
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Further ReadingYou can view other papers from the 2011 Nebraska Swine Report by clicking here.Find out more information on PMWS by clicking here. |
September 2012