Optimal vaccination strategy against Mycoplasma hyopneumoniae, porcine reproductive and respiratory syndrome virus, and porcine circovirus type 2 in case of early M. hyopneumoniae infection

Abstract Background The aim of this study was to determine the optimal vaccination strategies for the control of porcine respiratory disease complex (PRDC) caused by Mycoplasma hyopneumoniae, porcine reproductive and respiratory syndrome virus (PRRSV), and porcine circovirus type 2 (PCV2) in case of early mycoplasmal infection. Methods A total of 120 pigs were randomly divided into 6 groups (20 pigs per group). Four separate vaccine regimen groups were selected. Pigs from the four vaccinated groups were challenged with M. hyopneumoniae at 28 days old followed by a challenge of PRRSV or PCV2 at 49 days old. Results Regardless of PRRSV or PCV2 vaccination, pigs vaccinated with one of the M. hyopneumoniae vaccines at 7 days old had a significantly better growth performance over the whole length of the study compared to pigs vaccinated with a second M. hyopneumoniae vaccine at 21 days old. Vaccination of pigs with M. hyopneumoniae at 7 days and PRRSV at either 7, 14 or 21 days old resulted in significantly reduced PRRSV viremia and lung lesions compared to vaccination of pigs with M. hyopneumoniae and PRRSV at 21 days old. Conclusions The efficacy of the PRRSV MLV vaccine is influenced by the different timing of M. hyopneumoniae vaccination whereas the efficacy of the PCV2 vaccine is not. This experiment study demonstrated that early vaccination with a M. hyopneumoniae vaccine should be the highest priority in order to control M. hyopneumoniae and PRRSV infection in cases of early M. hyopneumoniae infection.


| INTRODUC TI ON
Porcine respiratory disease complex (PRDC) is a major health concern causing devastating economic losses to the swine industry due to a decrease in growth rates, and an increase in mortality and treatment cost. PRDC is a multifactorial disease caused by a combination of infectious pathogens, environmental stressors and production system (Chae, 2016). Among the multiple aetiological agents, Mycoplasma hyopneumoniae, porcine reproductive and respiratory syndrome virus (PRRSV), and porcine circovirus type 2 (PCV2) are the three most common pathogens causing PRDC in Asian countries (Chae, 2016).
In the field, swine practitioners and producers use vaccination rather than antibiotic treatment and prefer to use single-dose vaccines against M. hyopneumoniae, PRRSV and PCV2 in order to control PRDC efficiently. Therefore, a vaccination regimen strategy based on the natural incidence of infection is extremely important in order to avoid animals becoming infected prior to vaccination. In Japanese field conditions, pigs typically become co-infected with PRRSV and PCV2 at around 49 days old. However, the age of mycoplasmal infection is getting younger based on early detection of M. hyopneumoniae DNA in laryngeal swabs collected from suckling piglets. Detection of M. hyopneumoniae in the tonsils and bronchus of suckling piglets is clinically significant because enzootic pneumonia caused by M. hyopneumoniae may start relatively early in the farrow-to-finish production system (Sibila et al., 2007), which is the most common production system in Japanese farms. Once weaned, these piglets are exposed to a higher mycoplasmal pressure because they come into contact with other litters of piglets increasing the risk of infection with M. hyopneumoniae. Vaccination against M. hyopneumoniae is typically administered at 21 days old. However, vaccination at 7 days of age may be necessary in order to avoid transmission of M. hyopneumoniae among the weaned piglets and to control enzootic pneumonia during the fattening period in farms where M. hyopneumoniae is detected in laryngeal swabs from suckling piglets. Although it has not been clearly shown which of these three pathogens (M. hyopneumoniae, PRRSV and PCV2) plays the biggest role in causing PRDC, controlling M. hyopneumoniae infection may be the most critical because previous studies have shown that M. hyopneumoniae is able to enhance the replication of PRRSV and PCV2, potentiating the severity of pneumonia in pigs (Opriessnig et al., 2004;Thacker, Halbur, Ross, Thanawongnuwech, & Thacker, 1999). Furthermore, a single-dose vaccination against M.
hyopneumoniae resulted in a reduction of PRRSV-induced lung lesions whereas a single-dose vaccination against PRRSV did not result in a reduction of M. hyopneumoniae-induced lung lesions in pigs that were dually infected with PRRSV and M. hyopneumoniae . To the best of our knowledge, no one has so far per-

| Inocula
The inocula used for challenge in this study were; Japanese PRRSV strain Jpn5-37 (North American genotype, lineage 1) which was isolated from a 9-week-old diseased pig on a farm with a high mortality rate (21%) during 2007(Iseki et al., 2016, M. hyopneumoniae strain SNU98703 and PCV2b strain SNUVR000463 (GenBank KF871068). Japanese PRRSV strain Jpn5-37 was kindly provided by Dr. Michihiro Takagi (Viral Research Team, National Institute of Animal Health, Tsukuba, Ibaraki, Japan). Co-infection with PCV2 strain SNUVR000463 and M. hyopneumoniae strain SNU98703 was reported to cause severe pneumonia in lungs and lymphoid depletion in the lymph node of the infected pigs ).

| Animals
A total of 120 colostrum-fed, cross-bred, conventional piglets were purchased at 7 days old from a commercial farm. Male piglets were castrated at 5 days of age. The farm was free of PRRSV and M. hyopneumoniae based on serological testing of the breeding herd, and long-term clinical and slaughter history. In addition, none of the sows were immunized against PRRSV, PCV2 and M. hyopneumoniae.

| Experimental design
A total of 120 pigs were randomly divided into 6 groups using the random number generation function (Excel, Microsoft Corporation; Figure 1). Each group contained 20 piglets, 10 were male and 10 were female. All the pigs were randomly assigned into 12 rooms.
At 0 dpc (49 days old), pigs in the VacA113/Ch, VacA123/Ch, VacA133/Ch, VacB333/Ch and UnVac/Ch groups were challenged with PRRSV and PCV2. First, they were intranasally inoculated with 3 ml of PRRSV virus stock containing 1.2 × 10 5 of 50% tissue culture infective dose (TCID 50 )/ml. Five hours after PRRSV inoculation, pigs were again intranasally administered 3 ml of PCV2 virus stock containing 1.2 × 10 5 TCID 50 /ml. The 5 hr interval between challenges was applied in order to avoid mixture of two pathogens and potentially a decrease in infectivity. Pigs in the UnVac/UnCh group were the negative control. (147 days old), and 126 (175 days old) dpc. Five pigs from each group were randomly selected for euthanasia and necropsy at 0 (49 days F I G U R E 1 Experimental design. Pigs were administered a M. hyopneumoniae (Mhp), a porcine reproductive and respiratory syndrome virus (PRRSV), and a porcine circovirus type 2 (PCV2) and challenged with M. hyopneumoniae, PRRSV, and PCV2 on certain days as shown. A number of pigs were necropsied as shown old) and 21 (70 days old) dpc. Ten pigs from each group were sedated by an intravenous injection of sodium pentobarbital and then euthanized by electrocution at 126 (175 days old) dpc as previously described (Beaver et al., 2001). Tissues were collected from each pig at necropsy. Tissues were fixed for 24 hr in 10% neutral buffered formalin, and embedded in paraffin.

| Clinical observation
Pigs were monitored daily for clinical signs and scored weekly using scores ranging from 0 (normal) to 6 (severe dyspnoea and abdominal breathing; Halbur et al., 1995). Observers were blinded to vaccination and type of vaccine status. Mortality rate was calculated as the number of pigs that died divided by the number of pigs initially assigned to that group within batch. Pigs that died or were culled throughout the study were necropsied.

| Quantification of PRRSV RNA in blood
Serum samples were collected from individual pigs and RNA was extracted and PRRSV genomic cDNA copy numbers were quantified, as previously described (Wilson et al., 2013). Real-time PCR was also performed for the PRRSV MLV vaccine viruses and challenge PRRSV to quantify their respective genomic cDNA copies Park, Seo, Han, Kang, & Chae, 2014;Wilson et al., 2013).

| Quantification of PCV2 DNA in blood
DNA was extracted from serum samples using a commercial DNA extraction kit (QIAamp DNA Mini Kit, QIAGEN). Real-time PCR was used to quantify PCV2 genomic DNA copy numbers (Gagnon et al., 2008).

| Serology
The serum samples collected from piglets were also tested for antibodies against PRRSV using IDEXX PRRS X3 Ab test (IDEXX

| Pathology
The severity of macroscopic lung lesions was given a score in order to estimate the percentage of the lung affected by pneumonia. The scoring was done by two pathologists (authors Oh and Chae) at the Seoul National University (Seoul, Republic of Korea).
For the entire lung (100 points) were assigned 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).
Lung and lymphoid tissue sections were blindly examined by two veterinary pathologists (Oh and Chae). Mycoplasmal pneumonia lesions were scored (0-6) based on the severity of peribronchiolar and perivascular lymphoid tissue hyperplasia (Thacker et al., 1999). Interstitial pneumonia lesions were scored (0-6) based on the severity of interstitial pneumonia as previously described (Halbur et al., 1995).

| Statistical analyses
Prior to statistical analysis, real-time PCR data were transformed to log 10 values. Data were tested for normal distribution using Shapiro-Wilk test. One-way analysis of variance (ANOVA) was performed for variables that showed normal distribution (ADWG, M. hyopneumoniae DNA, PRRSV RNA, PCV2 DNA, serology, macroscopic lung lesions and IFN-γ-SC). When a one-way ANOVA showed statistical significance, a pairwise test using Tukey's adjustment was performed to determine the significance of group differences at each time point. Kruskal-Wallis test was used with variable without a normal distribution (respiratory sign and microscopic lung lesions).
When the non-parametric Kruskal-Wallis showed statistical significance, Mann-Whitney test was performed to determine the significance of group differences at each time point. A P value <0.05 was considered as statistical significance.

| Clinical observation
Clinical signs were recorded weekly and the mean respiratory scores were compared among the six different groups. Between

| Growth performance
No statistical difference was observed in the average body weight (±standard deviation) among the six groups at 7 days old which was the start of the experiment.

| Quantification of PRRSV RNA in blood
Serum samples from pigs in all six groups were tested for the pres-   (Figure 3b).

| Quantification of PCV2 DNA in blood
Serum samples were also tested for the presence of PCV2. Prior to challenge, all serum samples were negative for PCV2. Pigs from the VacA113/Ch, VacA123/Ch, VacA133/Ch and VacB333/Ch groups had a significantly (p < .05) reduced number of PCV2 genomic copies compared to the UnVac/Ch group between 7 and 126 dpc. No genomic copies of PCV2 were detected in pigs from the UnVac/ UnCh group throughout the experiment (Figure 3c).

F I G U R E 5 Frequency of interferon-γ secreting cells (IFN-γ-SC). Mean number of M. hyopneumoniae-specific IFN-γ-SC (A), PRRSV-specific IFN-γ-SC (B)
and PCV2-specific IFN-γ-SC (C) in the different groups: VacA113/Ch ( ), VacA123/Ch ( ), VacA133/Ch ( ), VacB333/Ch ( ), UnVac/Ch ( ) and UnVac/UnCh ( ) groups. Variation is expressed as the standard deviation. Different letters within a sampling point mean statistically significant differences (p < .05)  should be administered at a younger age than the current vaccination at 3 weeks old. However, unlike this study, piglets may have the maternally derived antibodies under common Japanese field conditions. An experimental challenge study showed that vaccination of pigs at 7 days old with the same M. hyopneumoniae vaccine used in this study, was effective in reducing lung lesions even in the presence of maternally derived antibodies at a titre considerably higher than what is typically seen in the field (Wilson et al., 2012(Wilson et al., , 2013. Therefore, efficacy of M. hyopneumoniae vaccines is not interfered with maternally derived antibodies.

| D ISCUSS I ON
These results indicate that the timing of vaccination against M.
hyopneumoniae should be determined based on the age of infection rather than the presence of maternally derived antibodies. In addition, the timing of PCV2 challenge mimics natural infection in Japanese field conditions. In general, PCV2 infection starts at the end of the nursery or at the beginning of fattening periods, however, is usually quite variable depending on the farm (Takahaki, M. hyopneumoniae vaccination at 7 days old resulted in higher reduction of lung lesions after challenge at 28 days old, compared to mycoplasma vaccination at 21 days old. A concern is that early vaccination may not be effective due to a lack of immune system maturity within the piglets resulting in decreased vaccine efficacy.
In study regarding to early M. hyopneumoniae vaccination, vaccination of pigs with M. hyopneumoniae at 7 days old has been shown to induce a stronger cell-mediated immune response compared to vaccination at 21 days old (Seo et al., 2013) suggesting a protective advantage of early vaccination. In addition, cell-mediated immunity as measured by activation of IFN-γ-SC plays an important role in protective immune response against M. hyopneumoniae (Thacker, Thacker, Kuhn, et al., 2000). Therefore, the pig immune system is unique in many ways (Butler, 2009;Sinkora et al., 2003) (Thacker et al., 1999;Thacker, Thacker, Young, & Halbur, 2000;Alstine, Stevenson, & Kanitz, 1996). In addition, it has also been reported that M. hyopneumoniae infection can impair the efficacy of a PRRSV MLV vaccine (Thanawongnuwech & Thacker, 2003   M. hyopneumoniae infection did not impair the induction of humoral and cell-mediated immunity by the PCV2 vaccine and vice versa . Therefore, the fact that there is only 1 week interval between M. hyopneumoniae vaccination and challenge does not affect the ability of the PCV2 vaccine to reduce PCV2 viremia. It is important to note that, in the present study, each single-dose vaccine of M. hyopenumoniae and PRRSV was administered at 7 days of age (VacA113/Ch group) into separate anatomical sites. It is possible that concurrent vaccination may interfere with immune responses to each vaccine. However, the immune responses elicited in this study by the concurrent administering of the M. hyopneumoniae and PRRSV vaccines do not appear to be significantly different from immune responses following single administering of each vaccine based on a previous study (Jeong et al., 2018). Therefore, we believe that concurrent vaccination

AUTH O R ' S CO NTR I B UTI O N
SY was involved in performance of the experimental trials, data analysis and writing of the manuscript. TO was involved in preparation of the inoculum and laboratory analysis. JM and AI were involved in data analysis and writing of the manuscript. CC was involved in development of protocol, design of the study, review of the final manuscript, approval for publication. All authors read and approved the final manuscript.

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.