A field efficacy trial of a trivalent vaccine containing porcine circovirus type 2a and 2b, and Mycoplasma hyopneumoniae in three herds

Abstract Background This field trial was designed to evaluate the efficacy of a new trivalent vaccine containing porcine circovirus type 2a and 2b (PCV2a/b), and Mycoplasma hyopneumoniae at three independent locations. Methods Three farms were selected based on their history of PCV2 and M. hyopneumoniae co‐infection. Each farm housed a total of 60, 3‐day‐old pigs that were randomly allocated to one of three treatment groups. Pigs were administered the trivalent vaccine intramuscularly with either a 1.0 ml dose at 3 and 24 days of age or a 2.0 ml dose at 21 days of age in accordance with the manufacturer's recommendations. Results Clinically, the average daily weight gain of the one‐dose and two‐dose vaccinated groups within all three farms was significantly higher (p < 0.05) than those of unvaccinated animals during the growing (70–112 days of age), finishing (112–175 days of age) and overall (3–175 days of age) stages of production. One‐dose and two‐dose vaccinated animals elicited neutralizing antibodies and interferon‐γ‐secreting cells (IFN‐γ‐SC), which reduced the amount of PCV2 in terms of blood load and reduced the severity of lymphoid lesions when compared with unvaccinated animals. Similarly, one‐dose and two‐dose vaccinated animals elicited IFN‐γ‐SC, which reduced the amount of M. hyopneumoniae in terms of laryngeal load and reduced the severity of lung lesions. Conclusions The intramuscular administration of either one or two doses of trivalent vaccine was not significantly different in any of the evaluated parameters. The results of field trial demonstrated that the trivalent vaccine was efficacious in the protection of swine herds where PCV2d and M. hyopneumoniae were in active circulation.

Mycoplasma hyopneumoniae lacks a cell wall, has a very small amount of genetic material and is one of the smallest bacteria in nature (Razin et al., 1998). Enzootic pneumonia, caused by M. hyopneumoniae, is one of the most prevalent diseases affecting swine production and inflicts significant economic losses due to the resulting reduced growth rate and feed conversion efficiency (Young et al., 2011). contains PCV2b, which is genetically close to PCV2d. Although PCV2abased vaccines may protect pigs against PCV2d Opriessnig, Gerber, Xiao, Mogler, et al., 2014;Opriessnig et al., 2017;Park et al., 2019), vaccine failure has also been reported in PCV2a-vaccined herds Ramos et al., 2015;Seo, Park, Kang, et al., 2014). The objective of this study was to determine the efficacy in relation to growth performance of a new trivalent vaccine containing PCV2a/b and M. hyopneumoniae in pig farms suffering from concurrent circulation of PCV2d and M. hyopneumoniae.

Farm history
The clinical field trial was conducted on three farms from June to December of 2020. Farms were labelled as 'A, B and C' and were 380-, 260-, and 430-sow, respectively, farrow-to-finish swine operations with an all-in-all-out production system. The status of porcine reproductive and respiratory syndrome virus (PRRSV) on all three farms was stable, with no active PRRSV circulation (high-parity sows were the only seropositive animals in the herd  (Chae, 2004;Segalés et al., 2005). A lung examination was performed at the slaughterhouse, and was suggestive of enzootic pneumonia with cranioventral bronchopneumonia lesions in 60% of the 30 pigs had. Farm C was selected based on its clinical history of PCVAD and enzootic pneumonia. Previous diagnoses fulfilled the definition of PCVAD (Chae, 2004) to include clinical signs (i.e. retardation of growth), histopathological findings (i.e. lymphoid depletion and lymphoid granulomatous inflammation with intracytoplasmic inclusion bodies), along with PCV2 antigen presence in lymphoid lesions as determined by immunohistochemistry in four out of five suspected animals on the farm. PCV2d was detected in serum from three pigs that ranged from 4.35 to 5.18 log 10 DNA copies/ml, which was consistent with defined PCVAD (Darwich et al., 2008;Segalés et al., 2005). A lung examination was performed at the slaughterhouse, which confirmed that 20% of the 30 pigs had mycoplasmal pneumonia lesions.

Study design
The results of this field study will be sent for registration and therefore groups were injected intramuscularly in the right side of the neck at study days 0 (3 days of age) and 21 (24 days of age) with 1.0 ml of the trivalent vaccine. Pigs in the UnVacA, UnVacB and UnVacC groups were injected intramuscularly in the right side of the neck at study days 0 (3 days of age) and 21 (24 days of age) with 1.0 ml of phosphatebuffered saline (PBS; 0.01 M, pH 7.4).
At 28 days of age, pigs from the vaccinated and unvaccinated groups were commingled and randomly assigned into six pens (10 pigs per pen) using the random number generator function (Excel, Microsoft Corporation). All pens were identical in design with equipment including free access to water and feed. Five pigs from each group were randomly selected and euthanized for necropsy at 112 days of age. The rest of pigs from each group were euthanized for necropsy at 175 days of age.
Pigs were sedated by an intravenous injection of sodium pentobarbital and then euthanized by electrocution as previously described (Beaver et al., 2001). Lung, liver, tonsil, kidney, spleen, small and large intestine and superficial inguinal lymph node tissues were collected from each pig at the time of necropsy. Tissues were fixed for 24 h in 10% neutral buffered formalin, routinely processed and embedded in paraffin. The protocol for this field study was approved by the Seoul National University Institutional Animal Care and Use Committee (approval number SNU-191017-10).

Sampling collection
Blood and laryngeal swabs were collected at study days 0 (3 days of age), 18 (21 days of age), 46 (49 days of age), 67 (70 days of age) and 109 (112 days of age). Pigs were snared and restrained with a mouth gag for laryngeal swab collection. Swabs were guided with a laryngoscope down into the larynx. The internal walls of the laryngeal cartilages were then swept with the swabs once the larynx was visualized and the epiglottis was in a low position as previously described (Pieters et al., 2017).

Mortality
Pigs that died were subjected to gross pathological examination within 24 h at local veterinary practitioners. All major organs such as brain, lung, superficial inguinal lymph node, small and large intestine, liver, kidney and tonsils were collected from each pig submitted to the diagnostic laboratory. Polymerase chain reaction assays were used in order to detect specific nucleic acids for PCV2, PRRSV, swine influenza virus and M. hyopneumoniae (Cai et al., 2007;Chung et al., 2002;Kim & Chae, 2004a;Lee et al., 2008). All other bacterial isolation and identifications were carried out by using routine methods.

Clinical observations
Pig physical condition was monitored daily, and pigs were scored weekly for clinical signs as previously described . Briefly, scoring was defined as follows: 0 (normal), 1 (rough haircoat), 2 (rough haircoat and dyspnoea), 4 (severe dyspnoea and abdominal breathing), 5 (severe dyspnoea and abdominal breathing, and hesitation of movement) and 6 (death). Scoring observers were blinded to vaccination status.

Growth performance
Pigs were weighed at study days 0 (3 days of age), 18 (21 days of age), 67 (70 days of age), 109 (112 days of age) and 172 (175 days of age). ADG (g/pig/day) was determined for study days 0-18, 18-67, 67-109 and 109-172. The ADG during these various 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.

PCV2 DNA in blood
A commercial kit (QIAamp DNA Mini Kit, QIAGEN, Valencia, CA, USA) was used to extract DNA from serum samples for PCV2d. The number of genomic DNA copies for PCV2a, PCV2b and PCV2d was then quantified by real-time PCR (Gagnon et al., 2008;Jeong et al., 2015). To construct a standard curve, real-time PCR was performed in quadruplicate in two different assays: (i) 10-fold serial dilutions of the PCV2 plasmid were used as the standard, with concentrations ranging from 10 10 to 10 2 copies/ml, and (ii) 10-fold serial dilutions of PCV2 cultured in PCV1-free PK-15 cells were used at concentrations ranging from 10 4.5 TCID 50 /ml to 10 −3.5 TCID 50 /ml. The PCV2 plasmid was prepared as described previously (Gagnon et al., 2008). Culture supernatants of PCV1-free PK-15 cells were used as negative control.

Mycoplasma hyopneumoniae DNA in laryngeal swabs
DNA was extracted from laryngeal swabs using the commercial kit  (Dubosson et al., 2004).
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 considered to be approximately one genome equivalent (Kurth et al., 2002). A positive and negative control was included in each run using chromosomal DNA from M. hyopneumoniae strain SNU98703 and double distilled water, respectively, as the template.

Serology
The presence of PCV2 and M. hyopneumoniae antibodies was evalu- Serum samples were tested for serum virus neutralization using PCV2d strain (SNUVR202002, GenBank no. MW821481) (Fort et al., 2007;Pogranichnyy et al., 2000). Serum samples were heat-inactivated at 56 • C for 30 min prior to performing the test. The neutralization titre with this assay was calculated as the reciprocal of the highest dilution of the serum that was able to 80% block PCV2 infection in PK-15 cells.
Thus, the lowest dilution contained 25% serum (1:1 dilution of serum + equal volume of PCV2d stock), thereby the detection limit of this assay was 2 log 2 .
The spots on the membranes were read by an automated ELISpot reader (AID ELISpot Reader, AID GmbH, Strassberg, Germany). The results were expressed as the number of responding cells/million PBMC.

Pathology
Two pathologists at the Seoul National University scored the severity of macroscopic lung lesions in order to estimate the percentage of the lung affected by pneumonia (Halbur et al., 1995;Opriessnig et al., 2004). Two blinded veterinary pathologists then examined the collected pulmonary and lymphoid tissue sections. Pulmonary lesions were scored the severity of peribronchiolar lymphoid tissue hyperplasia by mycoplasmal pneumonia lesions ranging from 0 to 6 (0, normal; 1, mild multifocal; 2, mild diffuse; 3, moderate multifocal; 4, moderate diffuse; 5, severe multifocal; 6, severe diffuse) (Opriessnig et al., 2004).
Severity of lymphoid lesion severity was scored from 0 to 5 (0, normal;

Immunohistochemistry
Immunohistochemistry for PCV2 was performed as previously described (Park et al., 2013). Nine sections (three sections from three different blocks) of the same lymph node of each pig were used for the morphometric analyses of immunohistochemistry.
Quantitative data were analysed from the prepared immunohistochemistry slides using the NIH Image J 1.45s Program (http://imagej.nih.gov/ij/download.html). PCV2 analysis was conducted by the random selection of 10 microscopic areas, where 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
All real-time PCR data and neutralizing antibody titres were transformed to log 10 and log 2 , respectively, values prior to statistical analy- where a value of p < 0.05 was considered to be significant.

Mortality
The overall mortality rate is summarized in

Clinical signs
Vaccinated pigs (VacA1 and VacA2) from farm A had significantly lower (p < 0.05) clinical sign scores when compared with unvaccinated animals (UnVacA) at study days 60-74 (Figures 1a and 1b). Farm B vaccinates (VacB1 and VacB2) also had significantly lower (p < 0.05) clinical sign scores when compared with unvaccinated animals (UnVacB), but at study days 46-81 (Figures 1a and 1b). On farm C, vaccinated pigs (VacC1 and VacC2) had significantly lower (p < 0.05) clinical sign scores when compared with unvaccinated animals (UnVacC) at study days 39-116 (Figures 1a and 1b). A difference in respiratory signs was not observed between one-dose and two-dose vaccinated groups in any of the three farms.

Growth performance
The body weight of pigs at study days 0 (3 days of age) and 21 ( (Table 3). There were no significant differences in the ADG between one-dose and two-dose vaccinated groups on all farms.  Figures 2a and 2b).

PCV2 viremia
The one-dose and two-dose vaccinated pigs from farms A, B and C had comparable number of genomic copies of PCV2d DNA throughout the entire field trials with no significant farm-to-farm differences between the three sites. Genomic copies of PCV2a and PCV2b DNA were not detected in any pigs from three farms throughout the entire field study.
The one-dose and two-dose vaccinated pigs from three farms had comparable number of genomic copies of M. hyopneumoniae DNA in their laryngeal swabs throughout the entire field trials, and significant differences were not found between groups on the three farms.

Immune responses against PCV2
Two-dose vaccinated pigs (VacA2, VacB2 and VacC2) from three farms and VacC2) from three farms had a significantly higher (p < 0.05) number of PCV2d-specific IFN-γ-SC at study days 46, 67 and 109 when compared with unvaccinated pigs (Figure 3c).

DISCUSSION
The common sign of PCV2 and M. hyopneumoniae co-infection is growth retardation. Vaccination against these two pathogens is needed and widely used to improve pig growth performance. Therefore, growth Germany (Heißenberger et al., 2013) and Switzerland (Kurmann et al., 2011). Swine practitioners and producers are therefore aware of the costly impact that subclinical PCV2 infection has in swine herds. This  (Fort et al., 2008(Fort et al., , 2009Meerts et al., 2005Meerts et al., , 2006 Pathological evaluation was also critical in evaluating the protective index as lesion reduction is related to growth performance in both PCV2 and M. hyopneumoniae infection (Jensen et al., 2002;Maes et al., 1998;Martelli et al., 2011;Segalés et al., 2009) proven. Commercial farm pigs used as field trials such as this are continuously exposed and re-exposed to the prevalent field PCV2d and M. hyopneumoniae by horizontal transmission. Natural co-infections as well as other intrinsic and extrinsic factors also exacerbate disease in these less-controlled commercial settings. A true evaluation of the direct effect of vaccination on pathological outcomes would require a controlled experimental challenge study.
Piglets also face potential interference from maternally derived antibodies (MDA) present at the time of vaccination. In general, early vaccination against PCV2 and M. hyopneumoniae was proven as effective in piglets less than 1 week of age regardless of MDA presence (O'Neill et al., 2011;Wilson et al., 2012). This field study did not evaluate the effect of MDA on vaccine efficacy. Additional studies are necessary to explore this theory and ultimately determine the effect of MDA on trivalent vaccine efficacy under well-controlled experimental conditions.