Đánh giá hiệu quả vaccine pcv2 năm 2024

This study investigated if parenteral administration of a prototype adjuvanted vaccine against porcine circovirus type 2 (PCV2) could override maternally derived antibodies and induce acquired immunity in young piglets. Piglets with high levels of maternal PCV2 antibodies at 1 wk of age were randomly grouped into vaccinates and controls on the basis of body weight and inoculated with the vaccine or a control preparation twice, with an interval of 3 wk. Both groups were challenged 3 wk after the booster vaccination and euthanized 3 wk after challenge. The pigs were evaluated for clinical disease, histologic lesions in sections of gastric and left inguinal lymph nodes stained with hematoxylin and eosin, and the amount of PCV2 antigen in the lymph nodes by immunohistochemical study. The PCV2 antibody titers were monitored by competitive enzyme-linked immunosorbent assay throughout the experiment. The vaccinates showed significantly less decline (P < 0.05) in PCV2 antibody titers after the booster vaccination. Clinical disease did not develop in any of the piglets. The vaccinates and controls did not differ in either histologic lesions or amount of PCV2 antigen in the lymph nodes. This study demonstrated some evidence of priming of young piglets in the presence of maternal antibodies. Further studies are recommended to determine the optimum concentration of PCV2 antigen and a suitable adjuvant for the vaccine to achieve the full potential of the strategy of inducing acquired immunity in young piglets that have maternally derived antibodies.

Résumé

Cette étude visait à déterminer si l’administration parentérale d’un prototype de vaccin avec adjuvant dirigé contre le circovirus porcin de type 2 (PCV2) pouvait outrepasser les anticorps maternels et induire une immunité acquise chez les jeunes porcelets. Les porcelets avec des niveaux élevés d’anticorps maternels anti-PCV2 à 1 sem d’âge étaient regroupés de manière aléatoire en vaccinés et témoins basés sur le poids corporel et inoculés avec le vaccin ou une préparation témoin deux fois à un intervalle de trois semaines. Les deux groupes ont été soumis à une infection défi 3 sem après la vaccination de rappel et euthanasiés 3 sem après l’infection. Les porcs ont été évalués pour la présence de maladie clinique, de lésions histologiques dans des sections de noeuds lymphatiques gastriques et inguinal gauche colorés avec de l’hématoxyline et éosine, et la quantité d’antigène PCV2 dans les noeuds lymphatiques par étude immunohistochimique. Les titres d’anticorps anti-PCV2 ont été suivis par épreuve immuno-enzymatique compétitive tout au long de l’expérience. Les animaux vaccinés ont présenté une diminution significativement moindre (P < 0,05) des titres d’anticorps anti-PCV2 après le rappel de vaccin. La maladie clinique ne s’est développée chez aucun des porcelets. Les animaux vaccinés et les témoins n’ont pas différé quant aux lésions histologiques et à la quantité d’antigènes de PCV2 dans les noeuds lymphatiques. Cette étude a démontré quelques évidences d’amorçage de l’immunité chez les jeunes porcelets en présence d’anticorps maternels. Des études supplémentaires sont recommandées afin de déterminer la concentration optimale d’antigène de PCV2 et un adjuvant adéquat pour le vaccin dans le but d’atteindre le plein potentiel de la stratégie d’induire une immunité acquise chez les jeunes porcelets possédant des anticorps maternels.

(Traduit par Docteur Serge Messier)

Introduction

Porcine circovirus type 2 (PCV2) is a small, nonenveloped, single-stranded DNA virus (). It has been incriminated as a necessary cause of postweaning multisystemic wasting syndrome (PMWS) (). This disease, which has a high case-fatality rate (), typically develops in piglets between 8 and 16 wk of age. Clinical signs include progressive emaciation, dyspnea, and enlarged superficial lymph nodes. The virus is also associated with a number of other diseases, such as porcine dermatopathy and nephropathy syndrome (), reproductive disorders (), proliferative and necrotizing pneumonia (), and porcine respiratory disease complex ().

Recently, a sharp increase in PCV2-associated deaths was reported in Canada (,). The virus is stable () and ubiquitous in the swine population (), which makes eradication very difficult. Vaccination represents an attractive means to control endemic infection and has been shown to be effective in controlling PCV2-associated diseases in some studies (,). Several commercial PCV2 vaccines are available in North America. There are 2 recommended vaccination strategies: i) vaccinating sows and relying on passive transfer of maternal antibodies, and ii) vaccinating young piglets to induce an active immune response. The effectiveness of sow vaccination can be complicated by several factors. First, maternally derived antibodies (MDAs) may confer only partial protection against PCV2 and rotavirus (,). Second, MDAs against PCV2 are short-lived (lasting 2 to 8 wk), vary markedly even among herd mates (), and would not last until the PMWS high-risk period, between 7 and 15 wk of age (,). Third, MDAs possess virus neutralization capacity (), but that alone may not be optimal or sufficient, since both humoral and cellular immunity may be essential in combating infection with this virus (). Finally, depending on the timing of infection, there may be no difference in mortality rates between piglets born to vaccinated sows and their nonvaccinated counterparts (). Vaccination of young piglets could be used as an alternative or together with sow vaccination; however, the efficacy of piglet vaccination may be negatively affected by MDAs against PCV2. To address the potential limiting effects of MDAs on active immunization, we investigated if parenteral administration of a prototype adjuvanted PCV2 vaccine to piglets at an early age could override MDAs against PCV2 and induce acquired immunity in young piglets.

Materials and methods

Preparation of vaccine antigen and formulation of the vaccine

The PCV2 vaccine antigen (Stoon 1010, PCV2a) was prepared in a collaborating laboratory according to previously reported methods (,). Briefly, the virus was propagated in PK-15 cells and subjected to 3 freeze-and-thaw cycles followed by sonication. The resulting cell lysate was centrifuged at 10 000 × g for 30 min at 4°C. The supernatant was collected and centrifuged at 70 000 × g for 4 h at 4°C. Virus pellets were harvested and resuspended in 1 mL of phosphate-buffered saline (PBS) at pH 7.2. The partially purified virus preparation then underwent sucrose gradient centrifugation, which yielded a high-titer pool. Next, β-propiolactone was added at 1:500 (v/v) and incubated for 6 h at 4°C, then added again at 1:1000 (v/v) and incubated for 18 h at 4°C to inactivate the virus particles. Complete inactivation was confirmed by titration for the infectivity in inoculated cell cultures by indirect immunofluorescence assay (). The concentration of virus antigen in the vaccine preparation was equivalent to 9.5 × 105 50% tissue culture infective doses (TCID50)/mL, as measured by antigen-capture enzyme-linked immunosorbent assay (ELISA) (,). The total protein concentration was 2.615 mg/mL, as measured by the Bradford method (). The LR4-incomplete oil-in-water adjuvant (Mérial, Athens, Georgia, USA) was heated at 37°C for 10 min and then stirred for 1 min before it was cooled rapidly to 4°C. The vaccine was diluted 1:1 with PBS (0.0036 M KCl, 0.0014 M KH2PO4, 0.136 M NaCl, 0.004 M Na2HPO4; pH 7.8) and maintained at 4°C until dispensed over 1 min into an equal amount of adjuvant that was being stirred slowly at 4°C. This vaccine formulation was stirred for an additional 1 min and stored at 4°C overnight. Similarly adjuvanted PCV-free PK-15 cell lysate served as the control formulation.

Preparation of challenge inocula

The challenge inocula consisted of tissue homogenate from the spleen of a gnotobiotic pig experimentally infected with PCV2a (Stoon 1010) and a lysate of PCV2a propagated in PK-15 cells, to be administered as loading and maintenance doses, respectively.

For the initial challenge a 10% (w/v) splenic tissue homogenate was sonicated for 30 s at full power and then centrifuged at 18 547 × g for 15 min at room temperature (RT). Chloroform (5% v/v) was added to the supernatant and mixed by constant inversion of the tube for 10 min at room temperature. The contents were centrifuged at 515 × g for 5 min at RT, and the top layer was removed from the chloroform and centrifuged at 92 736 × g for 4 h at 4°C. The pellets were suspended in PBS and filtered through a 0.2-μm filter. The virus titer in this inoculum was 4.58 × 105 TCID50/mL. For the second challenge, PCV2a was propagated in PCV-free PK-15 cells as described elsewhere (,). The medium covering the cell layer in tissue culture flasks (35 mL per T175 flask) was removed, the cells were washed twice with PBS, and the medium was replaced with minimum essential medium (Invitrogen, Carlsbad, California, USA) without added fetal calf serum or antibiotics. The cells were then frozen and thawed 3 times, sonicated for 30 s at full power, and centrifuged at 12 880 × g for 15 min at 4°C. The supernatant was collected and mixed with commercially available palatable syrup (Aunt Jemima Original; Quaker Oats Company, Chicago, Illinois, USA). The virus titer in the tissue culture lysate was 1.9 × 103 TCID50/mL.

Experimental design

Twenty 1-wk-old piglets from 4 litters were purchased from a farm where PCV2 vaccination was not practised, identified individually, and randomly assigned to vaccinate (n = 10) and control (n = 10) groups on the basis of body weight. They were located in 4 farrowing crates at the farm when they received primary vaccination at 1 wk of age. The vaccinates were administered 200 μL of the adjuvanted inactivated-PCV2 vaccine intradermally with the use of an injector gun (Biojector 2000; Bioject, Tualatin, Oregon, USA). Each dose contained PCV2 antigen equivalent to 4.75 × 104 TCID50. The controls were injected with 200 μL of the adjuvanted uninfected PK-15 cell lysate. At 3 wk of age the piglets were taken to the Western College of Veterinary Medicine animal care facility in Saskatoon, Saskatchewan, group-housed in a pen 15.4 m2, fed a commercial medicated diet until 8 wk of age, and thereafter switched to nonmedicated pig grower ration. Water was supplied ad libitum. The vaccinates and controls were administered a booster dose of the vaccine and control preparations, respectively, at 4 wk of age.

Challenge was done 3 wk after the 2nd vaccination, initially by administering orally 2 mL of the PCV2 inoculum prepared from spleen tissue homogenate containing 1.6 × 105 TCID50 of PCV2 per pig and then, for the next 3 d, by feeding inocula containing PCV2-infected PK-15 cell lysate and freshly cultured Campylobacter coli and Helicobacter cerdos mixed with a commercially available syrup, which delivered 3.6 × 103 TCID50 of PCV2 per pig per day.

The pigs were monitored visually for general health and clinical signs of PMWS during the experiment and euthanized by captive bolt 3 wk after challenge. Gross lesions were assessed by a team of experts in this field, who carried out the postmortem examinations. Inguinal, axillary, bronchial, mesenteric, and gastric lymph nodes, as well as thymus, tonsils, spleen, liver, ileum, and bone marrow, were collected in 10% buffered formalin for histopathological study. Blood was collected before each vaccination, challenge, and euthanasia; i.e., at weeks 1, 4, 7, and 10.

The experimental protocol was approved by the University of Saskatchewan Animal Care and Supply Committee (protocol 20050029).

Antigen-capture ELISA

Antigen-capture ELISA was done as previously described (,) to measure the amount of PCV2 antigen in the vaccine preparation and the challenge inocula. A flat-bottomed 96-well ELISA plate (Immunolon 4HBX; Thermo Electron Corporation, Milford, Massachusetts, USA) was coated with a monoclonal antibody (mAb; 2B1 F190) against PCV2 1002 isolate () in volumes of 100 μL per well at a dilution of 1:1500 in coating buffer (0.05 M sodium carbonate; pH 9.2). The plate was incubated overnight at 4°C. The coating buffer was then removed and the plate washed 4 times with PBS + 0.05% Tween20 (PBST). The test samples and a standard PCV2 capture ELISA antigen (Stoon 1010, pool 28) with a known TCID50 were serially diluted (1:250, 1:500, etc.) in PBST, and 100-μL volumes of each dilution were added to duplicate wells. The plate was incubated for 2 h at 37°C. After a similar washing step, 100 μL per well of rabbit anti-PCV2 polyclonal antiserum was added at a dilution of 1:2500 in PBST, and the plate was incubated for 1 h at 37°C. The plate was washed 4 times with PBST, 100 μL per well of biotinylated goat anti-rabbit antibodies (Invitrogen) were added at a dilution of 1:4000 in PBST, and the plate was incubated at 37°C for 1 h. The plate was washed 4 times in PBST, and 100 μL per well of streptavidin–peroxidase conjugate (ABC reagent; Vector Laboratories, Burlingame, California, USA) diluted in PBST according to the manufacturer’s recommendation was added and the plate incubated at 37°C for 30 min. The plate was again washed 4 times in PBST, and 3.3′, 5.5′-tetramethylbenzidine (TMB) substrate (KPL, Gaithersburg, Maryland, USA) was added, 100 μL per well. The reaction was stopped by the addition of 50 μL of 1 M H2SO4 per well after 15 min of incubation at ambient temperature. The optical density of the solution in the wells was read at 450 nm. The PCV2 antigen concentration in the test samples was calculated in terms of TCID50 from a standard curve plotted with the use of the respective concentrations in the standard PCV2 antigen dilutions.

Competitive ELISA

A competitive ELISA (cELISA) was used to measure the level of anti-PCV2 antibodies in pig serum as previously described (). The ELISA plate was coated with PCV2 antigen (PCV2 isolate 48285), 100 μL per well, at a dilution of 1:200 in a coating buffer (0.05 M sodium carbonate; pH 9.2). The plate was incubated overnight at 4°C. The coating buffer was then removed and the plates were washed 4 times with PBST. Each serum sample diluted at 1:125 in PBST was added in volumes of 50 μL per well in quadruplicate (2 adjacent rows and columns of the plate per sample) and incubated at 37°C for 45 min. Each plate included 4 wells each for blank (no serum: PBST only), negative, and positive control serum samples. In alternate columns (2, 4, 6, etc.) monoclonal antibody (B12B6) was added to the PCV2 at 1:100 in PBST in volumes of 50 μL per well. In the remaining columns (1, 3, 5, etc.) only PBST, in volumes of 50 μL per well, was added. The plates were incubated at 37°C for 30 min and washed 4 times with PBST. Then goat anti-mouse antiserum conjugated with horseradish peroxidase (Invitrogen) was added at a dilution of 1:5000 in PBST containing 5% (v/v) goat serum (Invitrogen), 100 μL per well. The plate was incubated at 37°C for 1 h and washed 4 times with PBST by the addition of 1M H2SO4, 50 μL per well, after incubation for 10 min at room temperature. The optical density of the substrate solution was read at 450 nm. Blank wells containing only the mAb without test serum represented a complete reaction (0% inhibition), whereas in wells containing both mAb and test serum containing PCV2 antibodies there is competition for the coated PCV2 antigen and the reaction is inhibited. The percentage inhibition was calculated for each sample as previously described ().

Histologic and immunohistochemical (IHC) studies

Among the 11 tissues collected, only inguinal and gastric lymph nodes were selected for further investigation owing to the lack of gross lesions observed at postmortem examination. The lymph nodes were fixed in 10% buffered formalin, embedded in paraffin, and sectioned at 5 μm. To detect PCV2 antigen, IHC was used with rabbit anti-PCV2 polyclonal antiserum (). Sections of left inguinal and gastric lymph nodes stained with hematoxylin and eosin were examined for lymphoid depletion, lymphoid hyperplasia (follicle formation), germinal center development, histiocytic hyperplasia, and syncytial giant cell formation. The amount of PCV2 antigen detected by IHC was evaluated from the intensity of staining, without knowledge of the sample’s identity, on a scale of 0 to 3+: 0 — negative; 1+ — minimal; 2+ — moderate; and 3+ — extensive.

Statistical analysis

The net change in cELISA values for individual pigs from primary vaccination to booster, challenge, and 3 wk after challenge was calculated by subtracting relevant individual-animal cELISA values. The Wilcoxon rank sum test was used to test comparisons, as the data were not normally distributed. Differences with a P-value < 0.05 were considered significant. Statistical testing was done with a commercial software package (Statistix 8; Analytical Software, Tallahassee, Florida, USA).

Results

Although the vaccination trial began with 10 pigs per group, 1 control pig was euthanized because of infectious arthritis, and data for another control pig were not considered because the pig was mistakenly vaccinated at the booster stage of the experiment. Therefore, the anti-PCV2 antibodies measured by cELISA that are presented in Figure 1 are for only 8 control pigs. The PCV2 MDA levels did not differ significantly between the 2 groups at 1 wk of age: there was more than 80% inhibition in all pigs. The antibody levels declined in the control pigs until challenge at 7 wk of age, whereupon the levels increased in all piglets except for no. 14.


Results of competitive enzyme-linked immunosorbent assay (cELISA) of antibodies to porcine circovirus-2 (PCV-2) in control pigs that twice received PCV-2-uninfected PK-15 cell lysate mixed 1:1 with an adjuvant and then were challenged orally with PCV-2 inocula.

Among the vaccinated piglets there were 2 patterns of change in PCV2 antibodies (Figure 2): 5 of the 10 pigs (subgroup A) maintained a higher level of antibodies (70% to 100% inhibition; P = 0.008) from week 1 to week 4 after primary vaccination, whereas in the remaining 5 pigs (subgroup B) the antibody levels decayed in a pattern similar to that of the controls. Only 3 of the pigs in subgroup A (nos. 13, 28, and 30) maintained a high level of immunity after the booster vaccination at 7 wk of age, and 1 of these pigs (no. 28) showed a decline in titer by week 10.


Results of the same cELISA in vaccinates that twice received inactivated PCV-2-infected PK-15 cell lysate mixed 1:1 with an adjuvant and then were challenged orally with PCV-2 inocula.

The cELISA values for the vaccinates were not significantly different from those for the controls at any time. All but 2 vaccinates (nos. 18 and 28) mounted an immune response to challenge (at week 10); these responsive pigs included 2 of the 3 vaccinates (nos. 13 and 30) that maintained high cELISA titers (~ 80% inhibition) at 7 wk of age. The net decline in cELISA values between weeks 1 and 7 was less in the vaccinates than in the controls (P = 0.04; Figure 3).


Box and whisker plot for the net change in cELISA values in controls and vaccinates after primary vaccination (week 1 to week 4), booster vaccination (week 1 to week 7), and challenge (week 1 to week 10). Outliers are indicated by diamonds. There was significantly (P < 0.05) less decline in the values in the vaccinates compared with those in the controls after booster vaccination.

None of the controls or vaccinated pigs had clinical signs of PMWS during the experiment or gross lesions at postmortem examination. The controls and vaccinates had microscopic lesions, including lymphoid hyperplasia, germinal center development, and histiocytic hyperplasia, none had lymphoid depletion or syncytial giant cell formation, 2 microscopic lesions characteristic of clinical PMWS. By IHC staining, PCV2 antigen was detected in left inguinal and gastric lymph nodes in 1 and 2 controls, respectively (Table I); the staining intensity was 1+ in all 3. Among the 9 vaccinates assayed, only 1 had positive staining in the left inguinal lymph node, with a score of 2+, and 3 had scores of 1+, 2+, and 3+, respectively, in the gastric lymph node. This finding suggests that there is no difference between controls and vaccinates in the proportion positive for PCV2 antigen. However, 2 of the vaccinates (nos. 6 and 22) had a higher intensity of staining in both tissues compared with the controls.

Table I

Staining-intensity scores in immunohistochemical testing for the presence of Porcine circovirus type 2 antigen in left inguinal and gastric lymph nodes

Lymph node Number with antigen staininga

Pig IDGroupLeft inguinalGastric3Control005Control007Control009Control01+14Control0021Control0024Control0026Control1+1+2Vaccinate004Vaccinate006Vaccinate02+10Vaccinate0013Vaccinate0018Vaccinate0019Vaccinate0022Vaccinate2+3+30Vaccinate01+

Discussion

As expected, given the endemicity of PCV2 infection in commercial swine herds (,), the controls and the vaccinated piglets had equally high levels of MDAs against PCV2 before vaccination. Although individual animals’ responses to vaccination can vary (), the reason for the 2 patterns of PCV2 antibody levels in the vaccinated group (subgroups A and B) after primary vaccination is not clear. To evaluate the immune response to vaccination in the pigs with MDAs, we compared the net change in cELISA values in both vaccinates and controls at week 7, after the booster vaccination. The decline in cELISA values in the vaccinates was significantly less than the decline in the controls, indicating an apparent immune stimulatory effect of vaccination. This effect might have been more pronounced with end-point titration, which is not a feature of this assay method.

The aim of this study was to investigate the efficacy of a parenterally administered PCV2 vaccine in the presence of MDAs against PCV2. Possible reasons for the absence of any remarkable effect of vaccination on the PCV2 antibody titers include the inhibitory effect of high concentrations of MDAs and/or an inadequate amount of antigen in the final vaccine preparation. Mechanisms by which high levels of MDAs can influence the efficacy of live attenuated vaccines include neutralization of the vaccine antigens and formation of complexes consisting of MDAs and the vaccine antigens that could mask antigen-specific epitopes, inhibition of B cell activation, and enhanced elimination by phagocyte cells (,–). Since an inactivated vaccine was tested in this study, formation of such complexes is a more likely reason for the retarded vaccine effect. Whereas MDAs are known to interfere with the humoral response, T cell priming may be unaffected (), although cell-mediated immune responses after the challenge were not examined in this study to provide evidence for this effect. A key determinant of MDAs’ interference is the ratio of vaccine antigen to MDAs; therefore, increasing the vaccine antigen dose might circumvent this issue ().

Both controls and vaccinates, including the pigs with high anti-PCV2 antibody titers, responded to the challenge inocula, which contained more PCV2 antigen than was in the vaccine. This observation demonstrated that the challenge dose was sufficient to induce an immune response and that the inhibition of immune induction by pre-existing antibodies is likely reversible by modification of the antigen:antibody ratio, which suggests that improved immune response may be achieved with increased antigenic mass in the vaccine. Piglets vaccinated at 4 and 7 wk of age with a recombinant pseudorabies virus expressing PCV2 capsid protein at a dose of 1 × 105 TCID50 of PCV2 antigen per pig (compared with the dose of 4.75 × 104 TCID50 per pig used in this study in 1-week-old piglets) had a significant anti-PCV2 immune response (). Another study examining the influence of MDAs on the efficacy of a PCV2 vaccine using 4-week-old piglets documented a significant reduction in microscopic lesions in lymphoid tissues and viremia in vaccinated pigs (). Together, these variable results suggest that the amount of MDAs as well as differences in vaccine formulation, including differences in adjuvants, may determine the success of priming for PCV2-specific immunity in piglets with passive immunity.

We conducted a parallel experiment with an additional group of pigs that were vaccinated against PCV2, C. coli and H. cerdos (data not shown) to study the influence of the latter 2 organisms on the development of gastric ulcers and PCV2-associated lesions. Hence, C. coli and H. cerdos were included in the challenge inocula in order to challenge simultaneously the 3 groups (controls, vaccinates receiving only PCV2, and vaccinates receiving PCV2 plus C. coli and H. cerdos) and to perhaps produce gastric inflammation to enhance the disease-producing potential of PCV2. Typical clinical signs or gross lesions of PMWS were not observed in either the controls or the vaccinates in this study, but the microscopic lesions reported in subclinical PCV2 infection () were seen in both groups. Further, the 2 groups were not different in IHC scoring. These observations indicate that although the challenge inocula produced an infection and induced microscopic lesions, there was no progression to clinical PMWS. The vaccine had little or no effect in either inducing a humoral response or preventing histologic lesions in vaccinates. A similar study evaluating the efficacy of a PCV2 vaccine with a different challenge model, an intranasally administered dose of 2 × 104.2 TCID50 per pig, found mild lesions with no significant differences between vaccinated and control pigs (). In the present study, a larger challenge dose and multiple days of challenge were used; there was an initial loading dose of 1.6 × 105 TCID50 per pig followed by a dose of 3.6 × 103 TCID50 per pig for the next 3 d. Infected tissue homogenates yield a high virus titer and have consistently been used as challenge inocula to reproduce clinical disease (,). Hence, we used a spleen tissue homogenate from an experimentally infected pig to provide a relatively large initial dose of PCV2. A PCV2 infection in PK-15 cells generally yields a low virus titer, never exceeding 1 × 105 TCID50/ml (). Therefore, we used a tissue culture lysate for the maintenance dose for 3 consecutive days. Previous PCV2 challenge studies have yielded variable results with regard to the production of disease (,–). In the present study it may be that previous exposure (or priming) or a protective concentration of MDAs precluded the development of clinical infection. Nevertheless, available study reports emphasize the frequent difficulty in modeling PCV2 infection in pigs randomly selected from commercial swine operations that would be candidates for PCV2 vaccination.

In conclusion, we examined the effects in young piglets of a prototypical inactivated adjuvanted vaccine in inducing active immunity against PCV2 and had limited success. Further studies using various vaccine-antigen doses in combination with different adjuvants administered to piglets with defined concentrations of MDAs are required to further address how to induce active immunity in young, passively immune pigs.

Acknowledgments

This study was supported by funding to Dr. John Ellis from the Natural Sciences and Engineering Research Council of Canada through the Special Research Opportunity program (grant PJ-5796306844). The authors are grateful to Dr. Allan Gordon at the Agri-Food and Biosciences Institute, Queen’s University Belfast, Belfast, Northern Ireland, for preparing and supplying the PCV2 vaccine antigen. We thank Carrie Rhodes and Crissie Auckland for technical support.