Efficacy of a novel bivalent vaccine containing porcine circovirus type 2d and Mycoplasma hyopneumoniae against a dual PCV2d and Mycoplasma hyopneumoniae challenge

Background Information on efficacy of a novel bivalent vaccine containing porcine circovirus type 2d (PCV2d) and Mycoplasma hyopneumoniae. Objective To evaluate bivalent vaccine for efficacy under experimental conditions. Animals Clinically healthy 35 weaned piglets at 18 days of age were used. Methods A 2.0 mL dose of bivalent vaccine was administered intramuscularly to pigs at 21 days of age in accordance with the manufacturer’s instructions. The pigs were challenged at 42 days of age either intranasally with PCV2d, or intratracheally with M. hyopneumoniae, or with both. Results Vaccinated-challenged pigs improved the growth performance compared to pigs that were unvaccinated and then, challenged. Vaccinated-challenged pigs elicited a significant amount of protective immunity for PCV2d-specific neutralizing antibodies and interferon-γ secreting cells (IFN-γ-SC) as well as for M. hyopneumoniae-specific IFN-γ-SC compared to unvaccinated/challenged pigs. Induction of systemic cellular and humoral immune responses from bivalent vaccination reduced the viral and mycoplasmal loads in the blood and larynx. Vaccination and challenge simultaneously reduced both lung and lymphoid lesion severity when compared to unvaccinated-challenged pigs. Discussion The results of this study demonstrated that the evaluated bivalent PCV2d and M. hyopneumoniae vaccine was efficacious in protecting pigs from the most predominant PCV2d genotype in the field today, as evaluated with a dual PCV2d and M. hyopneumoniae challenge under experimental conditions.


Introduction
Porcine circovirus 2 (PCV2) may best be described as a small but mighty virus. Postweaning multisystemic wasting syndrome (PMWS) was first reported in 1997 where the Western Canadian occurrence drew the attention of swine researchers and practitioners (1,2). PCV2 is the essential infectious causative agent of porcine circovirusassociated diseases (PCVAD) and is one of the most devastating and economically damaging pig diseases in the world. PCVAD denotes the entire spectrum of diseases that are associated with PCV2 including PMWS, porcine respiratory disease complex (PRDC), porcine dermatitis and nephropathy syndrome (PDNS), reproductive failure, and enteric disease (3,4).
Mycoplasma hyopneumoniae is the primary causative agent for enzootic pneumonia, a highly contagious respiratory disease in pigs (5,6). The disease causes large economical losses due to reduced growth performance, reduced feed efficiency, increased days to market, increased susceptibility to other respiratory infections, and increased medication cost (5,6).
The genetic gap between commercial PCV2 vaccines and the geographic prevalence of field viruses is increasing. All major commercially available PCV2 vaccines contain PCV2a and/or PCV2b while PCV2d is particularly predominant in the Asian and North American field (7)(8)(9)(10)(11)(12). PCV2d has proven as more virulent than PCV2a and PCV2b in co-infection models (13)(14)(15)(16). Moreover, PCV2d was repeatedly reported in association with perceived vaccine failures in several countries, indicating a "leaky" or imperfect vaccine situation (17)(18)(19). These field observations raise concern that commercially available PCV2a-based vaccines may not be fully capable of providing protection against the currently predominant PCV2d field genotype. Therefore, PCV2d-based vaccines may offer better protection against PCV2d infections compared to PCV2a-based vaccines. A new singledose bivalent vaccine containing inactivated whole PCV2d and M. hyopneumoniae has recently come onto the market. The objective of this study was to determine the efficacy of a bivalent vaccine containing PCV2d and M. hyopneumoniae against a dual experimental PCV2d and M. hyopneumoniae challenge.

Ethical statement
All of the methods were approved by the Seoul National University Institutional Animal Care and Use, and Ethics Committee (SNU-210308-2-1).

Experimental design
There were seven groups, each consisting of five pigs (two were male and three were female), for a total of 35 pigs and each group was segregated into a different chamber (Table 1). Each room was of the same size and design and provided the pigs free access to drinking water and feed a corn-soybean diet. The diet was a commercially available weaner diet that was formulated to provide 3,000 kcal/kg.  (21). Pigs in the Vac/ChP and UnVac/ChP groups only challenged with the PCV2d intranasally as described above, and pigs in the Vac/ChM and UnVac/ChM groups were only challenged with the same M. hyopneumoniae as explained above in the double challenged group sections. Pigs were sedated at 21 dpc (63 days of age) regardless of treatment by an intravenous injection of sodium pentobarbital prior to euthanization by electrocution as previously described (22). Tissues such as lung, subinguinal lymph node, tonsil, liver, heart, and intestine were collected from each pig at necropsy then fixed in a 10% neutral buffered formalin solution, before they were embedded in paraffin.

Clinical observation
Pigs were monitored daily and scored weekly for clinical signs by observers that were blinded to vaccination status. Scoring was defined according to the following scale: 0 (normal), 1 (rough haircoat), 2 (rough haircoat and mild dyspnea), 3 (mild dyspnea and abdominal breathing), 4 (moderate dyspnea and abdominal breathing), 5 (severe dyspnea and abdominal breathing), and 6 (death).

Average daily weight gain
Throughout the study, pigs were weighed at −21 (21 days of age), 0 (42 days of age), and 21 (63 days of age) dpc. Upon conclusion of the study, an average daily weight gain (ADWG = grams/pig/day) was calculated over three time points: (i) between −21 and 0 dpc, (ii) between 0 and 21 dpc, and (iii) between −21 and 21 dpc. ADWG was calculated by dividing the difference between the initial and final weights at each of the three experimental stages by the number of days in the period. Data were obtained in a blinded manner.

Quantification of PCV2d DNA
DNA was extracted from PCV2d serum samples with a commercial kit (QIAamp DNA Mini Kit, QIAGEN, Valencia, CA, United States). These extractions were then used to quantify the number of genomic DNA copies for both PCV2d (24). To construct a standard curve, real-time PCRd was performed in quadruplicate in 10-fold serial dilutions of the PCV2d DNA from PCV2d strain SNUVR202003, with concentrations ranging from 10 10 to 10 2 copies/ mL. The detection limit of the assay was 1.2 × 10 2 genomic copy numbers of PCV2d genotype (24). A negative control was included in each run using double distilled water as the template. Test was performed in duplicate. Data were obtained in a blinded manner.
Quantification of Mycoplasma hyopneumoniae DNA DNA was extracted from M. hyopneumoniae laryngeal swabs with a commercial kit (QIAamp DNA Mini Kit, QIAGEN, Valencia, CA, United States). These extractions were then used to quantify the number of genomic DNA copies for M. hyopneumoniae (25). Test was performed in duplicate. To construct a standard curve, real-time PCR was performed in quadruplicate in 10-fold serial dilution of chromosomal DNA from M. hyopneumoniae strain SNU98703, with concentrations ranging from 10 ng/μL to 1 fg/μL. One femtogram of chromosomal DNA from M. hyopneumoniae is approximately one genome equivalent (26). A negative control was included in each run using double distilled water as the template. Test was performed in duplicate. Data were obtained in a blinded manner.

Enzyme-linked immunosorbent assay
Antibody presence to both PCV2 and M. hyopneumoniae were tested in serum samples using two commercially available enzymelinked immunosorbent assay (ELISA) kits (INgezim CIRCO IgG

Serum neutralization test
PK-15 cells were planted into 96-well microtitration plates and used as an indicator for a serum viral neutralization assay (27,28). Each serum samples were serially diluted 2-fold up to 1:512. An equal volume of each sample dilution was mixed with equal volume of PCV2d stock at the titer of 200 TCID 50 /0.1 mL. Thus, the lowest dilution contained 25% (1:1 dilution + equal volume of PCV2 stock), thereby the detection limit of the assay was 2.0 log 2 . A positive and negative serum control from previous study was included in each run (29). Serum neutralization test was performed in duplicate. Data were obtained in a blinded manner.

Pathology
Macroscopic lung lesion severity was scored by two veterinary pathologists at the Seoul National University in order to estimate the percentage of the lung affected by pneumonia (31). To achieve this, lung and sections were collected and examined, then scored. Scoring was based on the severity of peribronchiolar lymphoid tissue hyperplasia by mycoplasmal pneumonia lesions on a scale of 0-6 (32). Real-time PCR confirmed that the lesions were a result of mycoplasmal pneumonia as previously described (25). Lymphoid lesion severity was scored (0-5) based on lymphoid depletion and granulomatous inflammation (33). Data were obtained in a blinded manner.

Immunohistochemistry
Porcine circovirus 2 immunohistochemistry was performed as previously described (34). Slides were prepared for immunohistochemistry morphometric analysis by sectioning off three cuts from three blocks of lymph node tissue per pig. Quantitative data was analyzed from the prepared immunohistochemistry slides using the NIH Image J 1.45 s Program. 1 Ten fields were selected at random for PCV2 analysis, and the number of the positive cells per unit area (0.25 mm 2 ) was determined as previously described (35) along with calculated mean values. Data were obtained in a blinded manner.

Statistical analysis
For statistical processing, real-time PCR data and neutralizing antibody titers were converted into decimal logarithmic and binary logarithmic values, respectively. A normal distribution was determined with the Shapiro-Wilk on these data. Whether or not the groups had statistically significant differences between them at various timepoints was then determined by performing a one-way ANOVA. For the further evaluation, a post hoc test for a pairwise comparison with Tukey's adjustment was conducted with a statistical significance result from a one-way ANOVA test. A Kruskal-Wallis test was additionally performed only in cases where the normality assumption was not met. Results which showed a statistical significance from the Kruskal-Wallis test were further evaluated with the Mann-Whitney test to compare the differences among the groups. Results were reported in p values and the values of p < 0.05 were considered significant.

Respiratory clinical outcomes
Within the dually challenged groups (PCV2d and M. hyopneumoniae), pigs in the Vac/ChPM group had significantly lower (p < 0.05) respiratory clinical scores than those in the UnVac/ ChPM group at 7, 14, and 21 dpc ( Figure 1A). A similar response was measured between the single PCV2d-challenged groups where pigs in the Vac/ChP group had significantly lower (p < 0.05) respiratory clinical scores than those in the UnVac/ChP group at 7 and 21 dpc ( Figure 1B). Responses from sole-M. hyopneumoniae challenge pigs followed suite, where pigs in the Vac/ChM group were significantly lower (p < 0.05) in respiratory clinical scores at 7 and 21 dpc compared with pigs in the UnVac/ChM groups ( Figure 1C). Respiratory clinical signs were not observed in the pigs in the UnVac/UnCh group throughout the entire experiment.

Growth performance
The body weight of the pigs was evaluated at −21 and 0 dpc, where significant differences between the seven groups were not found. Pigs in the Vac/ChPM that received a dual PCV2d and M. hyopneumoniae challenge and UnVac/UnCh groups had significantly higher (p < 0.05) body weights at 21 dpc than the pigs in the UnVac/ChPM group. A significantly higher (p < 0.05) ADWG was measured in pigs in the Vac/ChPM and UnVac/UnCh groups compared to pigs in the UnVac/ChPM group between 0 and 21 dpc. Pigs that received a single M. hyopneumoniae challenge, such as the Vac/ChM and UnVac/UnCh groups had significantly higher (p < 0.05) body weights compared to pigs in the UnVac/ChM group. Pigs in the Vac/ChM and UnVac/UnCh groups had a significantly higher (p < 0.05) ADWG than pigs in the UnVac/ChM group between 0 and 21 and between 0 and 21 dpc (Figure 2).

Genomic copies of PCV2d in serum
Serum was evaluated and determined negative of PCV2d genomic copies at both the time of vaccination and the time of PCV2d challenge. Vaccination and challenge significantly (p < 0.05) reduced the amount of PCV2d genomic copies found in the serum of pigs from the Vac/ChPM and Vac/ChP groups at 7, 14, and 21 dpc compared to unvaccinated-challenged pigs from the UnVac/ ChPM and UnVac/ChP groups (Figures 3A,B). Unvaccinatedunchallenged pigs remained free of PCV2d genomic copies as tested in the sera of the UnVac/UnCh group throughout the duration of the experiment.

Immunological responses against Mycoplasma hyopneumoniae
Vaccination and challenge (Vac/ChPM and Vac/ChM groups) resulted in significantly (p < 0.05) higher anti-M. hyopneumoniae antibody levels and M. hyopneumoniae-specific IFN-γ-SC levels as measured in Vac/ChPM and Vac/ChM group compared to the unvaccinated-challenged pigs (UnVac/ChPM and UnVac/ChM groups) at 0, 7, 14, and 21 dpc ( Figure 5). Anti-M. hyopneumoniae antibody levels and M. hyopneumoniae-specific IFN-γ-SC were not detected in the unvaccinated-unchallenged pigs from the negative control (UnVac/UnCh) group throughout the entire experiment.

Lung and lymphoid lesions
Vaccination and challenge (Vac/ChPM and Vac/ChM groups) resulted in significantly (p < 0.05) lower macroscopic and microscopic lung lesion scores compared to unvaccinated-challenged pigs (UnVac/ ChPM and UnVac/ChM groups) at 21 dpc. Macroscopic and microscopic lung lesions were not observed in the unvaccinated-unchallenged pigs from the negative control (UnVac/UnCh) group (Table 2).

PCV2 antigen in lymph nodes
Vaccination and challenge (Vac/ChPM and Vac/ChP groups) significantly (p < 0.05) lowered the number of PCV2 antigen Frontiers in Veterinary Science 07 frontiersin.org positive-cells in pigs lymph nodes compared to unvaccinatedchallenged pigs (UnVac/ChPM and UnVac/ChP groups) at 21 dpc. PCV2 antigen was not detected in the lymph nodes of the unvaccinated-unchallenged pigs from the negative control (UnVac/ UnCh) group (Table 2).

Discussion
A new bivalent vaccine containing PCV2d and M. hyopneumoniae provided good protection against an experimental dual PCV2d and M. hyopneumoniae challenge as observed by improved growth performance in vaccinatedchallenged pigs (Vac/ChPM and Vac/ChM groups) compared to unvaccinated-challenged pigs. Unlike the other vaccinatedchallenged groups, pigs vaccinated and then challenged with only PCV2d (Vac/ChP) did not significantly differ in growth performance from their unvaccinated-challenged counterparts (UnVac/ChP). As previously demonstrated, PCV2d alone is not enough to induce PCVAD experimentally, despite the fact that PCV2d is the primary causative agent of PCVAD (36).
Clinical outcomes of PCVAD are heavily dependent on PCV2 replication. High levels of PCV2 viremia led to clinical PCVAD with high mortality rates while low levels of PCV2 viremia led to a less obvious subclinical disease and reduced growth performance (37). This evaluation on PCV2 viremia is the primary indicator in the evaluation of PCV2 vaccine efficacy. The evaluated bivalent vaccine containing PCV2d and M. hyopneumoniae was able to significantly reduce PCV2d blood-viral load. Therefore, neutralizing antibodies against PCV2 and PCV2-specific IFN-γ-SC are considered protective immune responses as they play a key role in PCV2 clearance from both the blood and lymph nodes in swine as previous studies (38)(39)(40)(41)(42).
Porcine circovirus-associated disease is also characterized by lymphoid lesions such as granulomatous inflammation and lymphoid depletions (3,4). Lymphoid lesions were significantly reduced in vaccinated-challenged pigs. The evaluated bivalent vaccine containing Porcine circovirus 2 vaccination provided better overall protection against a homologous genotype rather than a heterologous genotype challenge (45-47). However, other studies show conflicting results (48,49). Results of PCV2d cross-protection by PCV2a vaccination based on levels of PCV2 viremia and PCV2-associated lesions are inconsistent. Several studies determined that PCV2a-based vaccines may produce a cross immune response to PCV2d and provide cross-protection against PCV2d infection (29, 46,49,50). These findings were contradicted in an additional study, where the evaluated PCV2a-based vaccine could not provide sufficient cross-protection against PCV2d infection based on immune responses (51). Nevertheless, our results should be interpreted cautiously because this study does not include heterologous vaccination and challenge groups, e.g., PCV2a vaccine against PCV2d challenge or  Thailand, and Vietnam (7,(9)(10)(11)(12). PCV2d is more virulent than the traditional PCV2a and PCV2b genotypes in pigs dual-infected with PRRSV or M. hyopneumoniae but not with PCV2 alone (13)(14)(15)(16)52). Therefore, a new bivalent vaccine containing PCV2d and M. hyopneumoniae may be useful candidate vaccine in swine herds to protect against PCV2d and M. hyopneumoniae infection.  Frontiers in Veterinary Science 10 frontiersin.org

Data availability statement
The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.

Ethics statement
The animal study was reviewed and approved by the Seoul National University Institutional Animal Care and Use, and Ethics Committee (SNU-210308-2-1).

Author contributions
SH: performance of the experimental trials and data analysis and writing of the manuscript. JS and TO: preparation of the inoculum and lab analysis. CK: making of the vaccine, design of the study, and writing of the manuscript. B-JS: making of the vaccine, design of the study, and writing of the manuscript. CC: development of protocol, design of the study, review of the final manuscript, and approval for publication. All authors contributed to the article and approved the submitted version.