Field evaluation of a sing‐dose bivalent vaccine of porcine circovirus type 2b and Mycoplasma hyopneumoniae

Abstract Background The field efficacy of a bivalent vaccine containing porcine circovirus type 2b (PCV2b) and Mycoplasma hyopneumoniae was evaluated on three pig farms. Methods Three pig farms were used, two of which had a history of subclinical PCV2 and clinical M. hyopneumoniae infections between 84 and 126 days of age while concurrent porcine circovirus‐associated disease and clinical M. hyopneumoniae infection between 70 and 105 days of age. Each farm vaccinated pigs with a single dose of a bivalent vaccine at 10 days of age while unvaccinated pigs were administered a single dose of phosphate buffered‐saline at the same age. Results Vaccination improved growth performance and reduced clinical scores significantly (p < .05) when compared with unvaccinated animals. The amount of PCV2d loads in blood and M. hyopneumoniae loads in nasal swabs of vaccinated animals were also significantly lower (p < .05) when compared with unvaccinated animals. Immunologically, vaccinated groups elicited a significantly higher (p < .05) level of protective immunity against PCV2d such as neutralizing antibodies and interferon‐γ secreting cells (IFN‐γ‐SC), as well as protective immunity against M. hyopneumoniae such as IFN‐γ‐SC when compared with unvaccinated animals. Pathologically, vaccination significantly lowered (p < .05) the scores of M. hyopneumoniae‐induced pneumonia and PCV2‐associated lymphoid lesions when compared with unvaccinated animals. Conclusions The evaluated bivalent vaccine provided good protection against PCV2d and M. hyopneumoniae infection under field conditions.


| INTRODUC TI ON
Porcine circovirus type 2 (PCV2) and Mycoplasma hyopneumoniae are two worldwide economically important dominating pathogens. PCV2 is a very small DNA virus which can be divided into at least eight different genotypes ('a' to 'h') based on its sequence identity in open reading frame 2 (ORF2) (Bao et al., 2018;Franzo & Segales, 2018;Wang et al., 2009;Xiao et al., 2015). Currently, PCV2d is the predominant genotype in Asia and North America (Franzo & Segales, 2018). PCV2 causes different clinical diseases and syndromes which are collectively referred to as porcine circovirus-associated disease (PCVAD). In Korea, PCV2 infection is so widespread that essentially all pig herds are infected with the pathogen. The decrease in PCVAD outbreaks since 2008 is attributed to the successful introduction of efficacious PCV2 vaccines to the market. Near or fully asymptomatic subclinical PCV2 infection remains the most common disease, leading to poor growth that increases days to market as described by other countries (Alarcon et al., 2013;Alarcon et al., 2013;Kurmann et al., 2011;Segalés, 2012).
Mycoplasmas including Mycoplasma hyopneumoniae are the smallest self-replicating bacteria and are distinguished by the lack of a cell wall that is present in most bacteria (Razin et al., 1998).
M. hyopneumoniae is the primary causative agent for enzootic pneumonia; one of the most widespread and costly diseases in the swine respiratory disease worldwide today. It is characterized by significant economic losses due to slower growth and poor feed conversion.
Coinfection with PCV2 and M. hyopneumoniae is one of the most devastating and damaging combinations of pig diseases.
Vaccination against PCV2 and M. hyopneumoniae is one of the most cost-effective strategies and is widely used in Asian pork production. A new bivalent vaccine containing PCV2b and M. hyopneumoniae (Circo/MycoGard, Pharmgate Animal Health, Wilmington, NC, USA) was first introduced into the Asian market. In particular, this bivalent vaccine is clinically interesting because it contains the PCV2b genotype. Genetically, PCV2b and PCV2d (initially called a mutant of PCV2b) are more closely related than PCV2a and PCV2d (Xiao et al., 2015).

| Farm history
The clinical field trial was conducted on three farms (A, B and C) between May and October of 2019. Farms A, B and C were each a 380-sow, 160-sow and 430-sow (respectively), farrowto-finish swine farm with an all-in-all-out production system.
All three farms are located in Chungcheongnam-do. Sows from these selected farms had not received vaccines against PCV2 or M. hyopneumoniae, while all piglets from this production system were immunized against both pathogens. Farms A and C were selected based on history of concurrent subclinical PCV2 infection and enzootic pneumonia. Herds approximately 12-18 weeks of age displayed subclinical PCV2 infection and enzootic pneumonia characterized by respiratory signs and growth retardation which were mainly associated with a marked increase in the mortality rate (approximately 7% and 5% of Farms A and C, respectively) from 21 to 140 days of age. Lungs were examined at the slaughterhouse where it was confirmed that 12 of 20 Farm A pigs and 15 of 20 Farm C pigs had mycoplasmal pneumonia lesions based on scoring from a previous method (Goodwin & Whittlestone, 1973).

| Clinical field study design
The experimental design for the field study strictly adhered to the guidelines set by QIA. QIA guidelines required that 20 piglets (male = 10 and female = 10) were selected and assigned to each group of vaccinated and unvaccinated animals. In an effort to minimize sow variation, four to six 7-day-old piglets were randomly selected from each sow and assigned evenly to either the vaccinated or unvaccinated group using the random number generation func- pigs were comingled before they were randomly distributed into four different pens kept within one room. Each pen contained 10 pigs with a similar proportion of each treatment per pen. Pens were identical in design and equipment which included free access to a feed and water trough. Standard farm procedures were followed regarding the feeding and watering of study animals. Blood and nasal swabs were collected at 0 (10 days of age), 18 (28 days of age), 39 (49 days of age), 81 (91 days of age) and 102 (112 days of age) days post-vaccination (dpv). All methods used in this study were approved by the Seoul National University Institutional Animal Care and Use Committee.
Observers were blinded to vaccination status.

| Growth performance
The live weight of each pig was measured at 10 (0 dpv), 70 (60 dpv), 112 (102 dpv) and 175 (165 dpv) days of age. The average daily weight gain (ADWG; gram/pig/day) was analysed over three time periods: (1) between 10 and 70 days of age, (2) between 70 and 112 days of age and (3) between 112 and 175 days of age. ADWG during the different production stages was calculated as the difference between the starting and final weight divided by the duration of the stage. Data for dead or removed pigs were included in the calculation.

| Quantification of PCV2d DNA in blood
DNA was extracted from serum samples by use of a commercial kit (QIAamp DNA Mini Kit, QIAGEN, Valencia, CA, USA). Real-time PCR was used to quantify the number of genomic DNA copies for PCV2d (Jeong et al., 2015).

| Quantification of M. hyopneumoniae in nasal swabs
DNA was extracted from nasal swabs by use of a commercial kit (QIAamp DNA Mini Kit, QIAGEN). Real-time PCR was used to quantify the number of genomic DNA copies for M. hyopneumoniae was quantified by real-time PCR (Dubosson et al., 2004).

| Serology
Enzyme-linked immunosorbent assay (ELISA) was used to test for both PCV2 and M. hyopneumoniae antibodies with commercial ELISA kits (SERELISA PCV2 Ab Mono Blocking, Synbiotics, Lyon, France, and M. hyo Ab test, IDEXX Laboratories Inc. Westbrook, ME, USA).
Serum samples were considered as positive for anti-PCV2 antibodies if the reciprocal ELISA titre was > 350 and as positive for M. hyopneumoniae antibody if the sample-to-positive (S/P) ratio was ≥ 0.4 in accordance with the manufacturer's instructions for each kit.

| Enzyme-linked immunospot assay
Enzyme-linked immunospot assay was used to measure the numbers of M. hyopneumoniae and PCV2d-specific interferon-γ secreting cells (IFN-γ-SC). Peripheral blood mononuclear cells (PBMC) were stimulated using the aforementioned challenge M. hyopneumoniae and PCV2d strains (Jeong et al., 2018) with results reported as the numbers of IFN-γ-SC per million PBMC.

| Pathology
The severity of macroscopic lung lesions was scored by two pathologists (Chae and one graduate student) at the Seoul National University (Seoul, Republic of Korea) to estimate the percentage of the lung affected by pneumonia. Scoring was performed out of 100 total possible points over the entire lung as follows: 10 points each to the right cranial lobe, right middle lobe, left cranial lobe and left middle lobe; 27.5 points each to the right caudal lobe and left caudal lobe and 5 points to the accessory lobe (Halbur et al., 1995).
Collected lung and lymphoid tissue sections were examined by two blinded veterinary pathologists (Chae and one graduate student). The severity of peribronchiolar and perivascular lymphoid tissue hyperplasia was assessed by scoring mycoplasmal pneumonia lesions (0 to 6) (Opriessnig et al., 2004). Mycoplasmal pneumonia lesions were confirmed by real-time PCR from lung lesions (Dubosson et al., 2004). The severity of lymphoid lesions was scored (0 to 5) based on the severity of lymphoid depletion and granulomatous inflammation (Kim & Chae, 2004).

| Immunohistochemistry
Immunohistochemistry for PCV2 was performed as previously described (Park et al., 2013). For the morphometric analyses of immunohistochemistry, three sections were cut from each of three blocks of tissue from lymph node of each pig. The slides were analysed using the NIH Image J 1.45s Program (http://imagej.nih.gov/ij/ downl oad.html) to obtain the quantitative data. For the analysis of PCV2, 10 fields were randomly selected, and the number of positive cells per unit area (0.95 mm 2 ) was determined as previously described (Kim et al., 2003). The mean values were also calculated.

| Statistical analysis
Prior to statistical analysis, real-time PCR and neutralizing antibody data were transformed to log 10 and log 2 values respectively. Data were tested for the normal distribution using the Shapiro-Wilk test and either the Student's t test or Mann-Whitney test were used to examine whether significant statistical differences existed between the two groups at each time point. The student's t test was conducted to compare the difference between the two groups, only if the normality assumption was met, while the Mann-Whitney test was performed to compare the differences between the two groups when the normality assumption was not met. A value of p < .05 was considered to be significant.

| Clinical evaluation
The vaccinated group on Farm A had the significantly lower (p < .05) clinical scores between 25 and 74 dpv when compared with the unvaccinated group. Clinical scores were significantly lower (p < .05) in the Farm B vaccinated group between 32 and 74 dpv, and at 95 and 102 dpv when compared with the unvaccinated group. Farm C clinical scores were significantly lower (p < .05) between 32 and 95 dpv in the vaccinated group when compared with the unvaccinated group ( Figure 1).

| Growth performance
A significant difference (p < .05) in the ADWG was observed on Farm A between vaccinated and unvaccinated group between 10 and 70, between 70 and 112 and between 112 and 175 days of age.
The ADWG in the Farm B vaccinated group during the 10-70 and 112-175 days period was significantly higher (p < .05) than that of the unvaccinated group. The overall growth performance of all three farms (10 to 175 days of age) of vaccinated groups was significantly higher (p < .05) than that of unvaccinated group (Table 1).

| Mortality
Farm A mortality included one unvaccinated pig that died of suppu-

| Quantification of PCV2d DNA in blood
PCV2 DNA was not detected in the blood of either vaccinated or unvaccinated animals at 0 and 18 dpv at any of the three farms.

| Immunological responses against PCV2
At the time of vaccination (10 days of age; 0 dpv), significant differences between vaccinates and non-vaccinates were not detected at any of the three farms in regard to anti-PCV2 antibodies. The PCV2 ELISA titres (Figure 3a) and PCV2-specific NA (Figure 3b) were significantly higher (p < .05) in the vaccinated group when compared with the unvaccinated group at 18-102 dpv in three farms. The mean frequencies of PCV2-specific IFN-γ-SC remained at basal levels (< 20 cells/10 6 PBMC) in both groups until 0 dpv. Thereafter, the mean number of PCV2-specific IFN-γ-SC was significantly higher (p < .05) TA B L E 1 Average daily weight gain (ADWG), mortality rate and pathology between vaccinated (Vac) and unvaccinated (UnVac) animals on three Farms in the vaccinated group when compared with the unvaccinated group from 18 to 81 dpv (Farms A and C) and from 18 to 102 dpv at Farm B (Figure 3c). and from 39 to 81 dpv (Farms B and C) (Figure 4b).

| Pathology
The results of lung and lymphoid lesion score are summarized in Table 1 Variation is expressed as the standard deviation. * Significant difference (p < .05) between vaccinated and unvaccinated group within the same farm the onset of active immunity has been proven to begin as early as in 5-day-old piglets, post-vaccination (O'Neill et al., 2011).
Vaccination at such a young age may result in additional complications, however, as the piglets face potential interference of maternally derived antibodies (MDA) present at the time of vaccination. The role of PCV2 MDA plays in active immunity of piglets after vaccination is a somewhat controversial issue. It has been stated that PCV2 MDA can affect the age of PCV2 infection (Rose et al., 2012) while contradictory evidence exists that demonstrated that high PCV2 MDA titres (≥ 10 log 2 ) could interfere with piglets' active seroconversion after vaccination (Fort at al., 2009).
In the present study, piglets with high ELISA (> 9 log 2 ) or NA (> 7 log 2 ) titres seemed to show interference with the development of the humoral immune response after vaccination. Most of the piglets (> 80% in three Farms) had lower ELISA (< 9 log 2 ) or NA (< 7 log 2 ) titres at the time of vaccination (data not shown  (Wilson et al., 2012(Wilson et al., , 2013. Moreover, protective immunity against M. hyopneumoniae is primarily cell mediated (Djordjevic et al., 1997;Thacker et al., 2000) and the present field trial proved that the bivalent vaccine elicited M. hyopneumoniae-specific IFN-γ-SC even in the presence of MDA. Therefore, a MDA effect on active immunization after vaccination is less likely to have an impact on the efficacy of the bivalent vaccine.
The bivalent vaccine is able to elicit protective immunity against PCV2 and M. hyopneumoniae. PCV2-specific NA and IFN-γ-SC are key needed component to reduce PCV2 viremia and lymphoid lesions (Chae, 2012;Fort at al., 2009;.

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interests with respect to their authorship.

E TH I C A L S TATEM ENT
All of the methods were previously approved by the Seoul National University Institutional Animal Care and Use, and Ethics Committee.
Sample collection was carried out according to the animal welfare code of Korea.

PE E R R E V I E W
The peer review history for this article is available at https://publo ns.com/publo n/10.1002/vms3.420.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.