Abstract
The objective of this study was to compare the efficacy of commercially available porcine circovirus type 2 (PCV2) and Mycoplasma hyopneumoniae vaccines. A total of 80 pigs was randomly divided into 6 treatment groups; 4 of the groups each received a different vaccine as well as a dual challenge. The remaining 2 groups were used as controls, 1 of which also received a dual challenge. Two of the 4 groups of pigs were administered 2 monovalent vaccines (designated as either monovalent vaccine A or B) of PCV2 at 7 days old and of M. hyopneumoniae at 21 days old. The remaining 2 vaccinated groups of pigs received a bivalent vaccine (designated as either bivalent vaccine A or B) of PCV2 and M. hyopneumoniae at 21 days old. All 4 vaccinated groups were challenged with M. hyopneumoniae at 42 days old [−14 d post-challenge (dpc)], followed by a PCV2d challenge at 56 days old (0 dpc). All 4 vaccinated/challenged groups displayed a reduction in clinical signs, PCV2d viremia, nasal shedding of M. hyopneumoniae, and lung lesions compared with pigs in the unvaccinated and challenged groups. Vaccination and challenge improved growth performance and increased the immunologic responses (M. hyopneumoniae- and PCV2-specific antibodies and interferon-γ-secreting cells) when compared to pigs in the unvaccinated/challenged groups. Pigs in groups vaccinated with either a monovalent or bivalent vaccine A treatment and challenge produced a larger amount of M. hyopneumoniae- and PCV2d-specific interferon-γ-secreting cells within the pigs and simultaneously reduced the nasal shedding of M. hyopneumoniae and PCV2d viremia compared with groups vaccinated with either a monovalent or bivalent vaccine B treatment and challenge. Both the bivalent vaccines and the respective monovalent vaccines were efficacious against a dual challenge of M. hyopneumoniae and PCV2d.
Résumé
L’objectif de la présente étude était de comparer l’efficacité de vaccins commercialement disponibles contre le circovirus porcin de type 2 (PCV2) et Mycoplasma hyopneumoniae. Un total de 80 porcs ont été divisés de manière aléatoire en six groupes de traitement; quatre des groupes ont chacun reçu un vaccin différent ainsi qu’une infection défi double. Les deux groupes restants ont servi de témoin, un des deux recevant l’infection défi double. Deux des quatre groupes de porcs ont reçu deux vaccins monovalents (désigné comme étant vaccin monovalent A ou B) de PCV2 à 7 jours d’âge et de M. hyopneumoniae à 21 jours d’âge. Les deux autres groupes de porcs vaccinés ont reçu un vaccin bivalent (désigné comme étant vaccin bivalent A ou B) de PCV2 et de M. hyopneumoniae à 21 jours d’âge. Les quatre groupes vaccinés furent challengés avec M. hyopneumoniae à 42 jours d’âge [−14 j post-défi (dpc)], suivi d’une infection défi avec PCV2 à 56 j d’âge (0 dpc). Les quatre groupes vaccinés/infectés ont montré une réduction des signes cliniques, de la virémie à PCV2d, de l’excrétion nasale de M. hyopneumoniae et de lésions pulmonaires comparativement aux porcs dans les groupes non-vaccinés et infectés. La vaccination et l’infection défi ont amélioré les performances de croissance et augmenté les réponses immunologiques (anticorps spécifiques contre M. hyopneumoniae et PCV2d et les cellules secrétant l’interféron-γ) lorsque comparé aux porcs dans les groupes non-vaccinés/infectés. Les porcs dans les groupes vaccinés avec soit le vaccin A monovalent ou bivalent et infectés ont produit de plus grandes quantités de cellules secrétant de l’interféron-γ spécifique à M. hyopneumoniae et PCV2d chez les porcs et ont simultanément réduit l’excrétion nasale de M. hyopneumoniae et la virémie de PCV2d comparativement aux groupes vaccinés avec le vaccin monovalent ou bivalent B et infectés. Autant les vaccins bivalents que les vaccins monovalents respectifs étaient efficaces à une infection défi double par M. hyopneumoniae et PCV2d.
(Traduit par Docteur Serge Messier)
Introduction
Porcine circovirus type 2 (PCV2) is the smallest known virus to autonomously replicate and causes a wide variety of syndromes that are collectively termed porcine circovirus associated disease (PCVAD) (1). At least 5 different genotypes have been identified to date and are designated with lowercase letters (a, b, c, d, and e). This alphanumerical ordering is based on the first identification of the virus (2). Among those, PCV2d has become the predominant genotype in North America and Asia, although PCV2a and PCV2b are still prevalent in these regions (3). Although PCV2 is the causative agent of PCVAD, subclinical infection remains the most common form of PCV2 infection worldwide (4).
Mycoplasma hyopneumoniae is the primary causative agent of enzootic pneumonia. It is considered one of the most economically important respiratory pathogens as it results in common and chronic disease in swine herds. Mycoplasma hyopneumoniae attaches itself to the ciliated epithelial cells lining the upper respiratory tract. As the bacterium colonizes, the cells are damaged, which predisposes the infected animals to secondary infection (5). Hydrogen peroxide and superoxide radicals are 2 toxic products of mycoplasmal metabolism that may cause further damage to the epithelial cells (6).
Porcine circovirus type 2 (PCV2) and M. hyopneumoniae are the 2 most prevalent and economically important pathogens in global pig production systems. These agents can have a significant negative impact on several areas of pig performance, such as weight gain and feed efficiency, particularly during the vital grow/finish phase of pig production. Co-infection with PCV2 and M. hyopneumoniae is commonly observed during the development of porcine respiratory disease complex (PRDC), which is responsible for major economic losses in the Asian pork industry.
Vaccination rather than administration of antimicrobial treatment is considered the more effective method to control pathogens associated with PRDC and is the most widely used. Vaccines against PCV2 and M. hyopneumoniae are the 2 most commonly administered vaccines in Korea, as in all Asian swine herds. Since the recommended vaccination time against both PCV2 and M. hyopneumoniae is similar, the use of a bivalent vaccine is preferred in an effort to reduce the number of injections that need to be administered. Despite this convenience, some swine practitioners and producers still prefer to use monovalent vaccines against the 2 pathogens. The objective of this study was to compare 2 different PCV2-M. hyopneumoniae bivalent vaccines with 2 sets of different PCV2 and M. hyopneumoniae monovalent vaccines against a dual M. hyopneumoniae and PCV2d challenge in swine.
Materials and methods
Animals
A total of 80, 7-day-old conventional piglets was purchased for this study. Piglets were colostrum-fed crossbreds and were selected from a commercial farm that was deemed free from porcine reproductive and respiratory syndrome virus (PRRSV) and M. hyopneumoniae based on serological testing of the breeding herd and long-term clinical and slaughter history. Piglets were tested for PCV2 and PRRSV viremia, as well as for nasal shedding of M. hyopneumoniae, all by real-time polymerase chain reaction (RT-PCR) upon arrival. Serology testing was also evaluated on the newly arrived piglets for PCV2 (SERELISA PCV2 Ab Mono Blocking; Synbiotics, Lyon, France), PRRSV (HerdCheck PRRS X3 Ab Test; IDEXX Laboratories, Westbrook, Maine, USA), and M. hyopneumoniae (M. hyo Ab Test; IDEXX Laboratories) antibodies. All piglets tested seronegative for PCV2, PRRSV, and M. hyopneumoniae.
Experimental design
The random number generator function (Excel; Microsoft Corporation, Redmond, Washington, USA) was used to randomly assign a total of 80 pigs into 6 groups (Figure 1). The following groups of pigs were then randomly assigned into 9 rooms: VacA-PM/Ch (n = 16, male = 8, female = 8); VacB-PM/Ch (n = 16, male = 8, female = 8); VacA-M+P/Ch (n = 16, male = 8, female = 8); VacB-M+P/Ch (n =16, male = 8, female = 8); and the unvaccinated/challenged group (UnVac/Ch) (n = 8, male = 4, female = 4). Pigs in the unvaccinated/unchallenged group (UnVac/UnCh) (n = 8, male = 4, female = 4) were randomly assigned into 1 room. Each room contained 2 pens with 4 pigs per pen. Individual pigs were also randomly assigned to pens using the random number generator function (Excel; Microsoft). All rooms and pens were uniform in design and equipment, including a freely accessible feed and water trough.
Figure 1.
Experimental design. Pigs were administered a vaccine against M. hyopneumoniae (Mhp) and/or porcine circovirus type 2 (PCV2) and challenged with M. hyopneumoniae and PCV2 on certain days as shown. A number of pigs were necropsied as shown.
Pigs in the VacA-PM/Ch group were administered a 2.0-mL dose of Fostera PCV MH (Serial No. 259044A; Zoetis, Parsippany, New Jersey, USA) intramuscularly at 21 d old. Pigs in the VacB-PM/Ch group were administered a 2.0-mL dose of Porcilis PCV M Hyo (Lot No. C530B01; MSD Animal Health, Boxmeer, Netherlands) intramuscularly at 21 d old.
Pigs in the VacA-M+P/Ch group were administered 2 monovalents: a 2.0-mL dose of RespiSure-One (Serial No. 263688; Zoetis) intramuscularly on the left side of the neck at 7 d old, followed by a 2.0-mL dose of Fostera PCV MetaStim (Serial No. 306218A; Zoetis) intramuscularly on the right side of the neck at 21 d old. Pigs in the VacB-M+P/Ch group were administered 2 different monovalent vaccines: a 2.0-mL dose of M+Pac (Serial No. 000511244; MSD Animal Health) intramuscularly on the left side of the neck, followed by a 2.0-mL dose of Porcilis PCV (Lot No. A545A01; MSD Animal Health) intramuscularly on the right side of the neck at 21 d old.
Pigs in the unvaccinated/challenged (UnVac/Ch) and unvaccinated/unchallenged (UnVac/UnCh) groups received a 2.0-mL dose of phosphate-buffered saline (PBS, 0.01M, pH 7.4) at 21 d old.
At −14 d post-challenge [(dpc), 42 d old], pigs in the VacA-PM/Ch, VacB-PM/Ch, VacA-M+P/Ch, VacB-M+P/Ch, and UnVac/Ch groups were anesthetized with a mixture of 2.2 mg/kg xylazine hydrochloride (Rumpon; Bayer Korea, Seoul, Korea) and 2.2 mg/kg tiletamine hydrochloride and 2.2 mg/kg zolazepam hydrochloride (Zoletil 50; Virbac Korea, Seoul, Korea) by intramuscular injection. Post-anesthetization, pigs were inoculated intratracheally with 7 mL of M. hyopneumoniae (strain SNU98703) culture medium containing 107 color-changing units (CCUs)/milliliter, as described in a previous study (7,8).
At 0 dpc (56 d old), pigs in the VacA-PM/Ch, VacB-PM/Ch, VacA-M+P/Ch, VacB-M+P/Ch, and UnVac/Ch groups were inoculated intranasally with 3 mL of tissue culture supernatant containing 1.2 × 105 TCID50/mL of PCV2d (SNUVR140004, GenBank no. KJ437506) (4).
Blood and nasal swabs were collected at the following timepoints: −49 dpc (7 d old); −35 dpc (21 d old); −14 dpc (42 d old); 0 dpc (56 d old), 7 dpc (63 d old); 14 dpc (70 d old); 35 dpc (91 d old); and 56 dpc (112 d old). At 175 d old (119 dpc), all pigs were sedated by an intravenous injection of sodium pentobarbital and then euthanized by electrocution, as described in a previous study (9). Animal methodology was approved by the Seoul National University Institutional Animal Care and Use Committee.
Clinical observations
The pigs were monitored daily for abnormal clinical signs and rated weekly using scores ranging from 0 (normal) to 6 (severe dyspnea and abdominal breathing) (10). Observers were blinded to type of vaccine status and vaccination. Pigs that died or were culled as deemed necessary were necropsied. At the end of the study, mortality rate was calculated as the number of pigs that died, divided by the number of pigs initially assigned to that group within the batch.
Growth performance
The live weight of each pig was measured at various timepoints throughout the study as follows: −35 dpc (21 d old); 0 dpc (56 d old); 28 dpc (84 d old); 56 dpc (112 d old); and 119 dpc (175 d old). At the end of the study, the average daily weight gain [(ADWG) grams/pig/day] was calculated over 4 time periods or production stages: i) from 21 to 56 d old; ii) from 56 to 84 d old; iii) from 84 to 112 d old; and iv) from 112 to 175 d old. During the different production stages, ADWG was calculated as the difference between the starting weight and final weight, divided by the number of days spanning the duration of the stage. Data for dead or removed pigs were included in the calculation.
Quantification of M. hyopneumoniae in nasal swabs
Real-time PCR was used to quantify the number of genomic deoxyribonucleic acid (DNA) copies for M. hyopneumoniae (11) once DNA was extracted from nasal swabs using a commercial kit (QIAamp DNA Mini Kit; QIAGEN, Valencia, California, USA).
Quantification of PCV2d DNA in blood
Real-time PCR was used to quantify the number of genomic DNA copies for PCV2d (12) once DNA was extracted from serum samples using the same commercial kit as for nasal swabs.
Enzyme-linked immunosorbent assay
Serum samples were tested for both M. hyopneumoniae and PCV2 antibodies using 2 commercially available enzyme-linked immunosorbent assay (ELISA) kits (M. hyo Ab Test; IDEXX Laboratories and SERELISA PCV2 Ab Mono Blocking; Synbiotics). Serum samples were considered positive for M. hyopneumoniae antibody if the sample-to-positive (S/P) ratio was ≥ 0.4 and positive for anti-PCV2 antibodies if the reciprocal ELISA titer was > 350, in accordance with the manufacturer’s instructions for each kit.
Enzyme-linked immunospot assay
The numbers of M. hyopneumoniae and PCV2d-specific interferon-γ-secreting cells (IFN-γ-SCs) were evaluated by enzyme-linked immunospot (ELISPOT) assay. Peripheral blood mononuclear cells (PBMCs) were stimulated using the challenge M. hyopneumoniae and PCV2d strains used in previous studies (12,13), with results reported as the numbers of IFN-γ-SCs per million PBMCs.
Pathology
The severity of macroscopic lung lesions was scored by 2 pathologists (Chae and a graduate student) at Seoul National University (Seoul, Republic of Korea) to estimate the percentage of the lung affected by pneumonia. Scoring was 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 (10).
Collected lung and lymphoid tissue sections were examined by 2 blinded veterinary pathologists (Chae and a graduate student). The severity of peribronchiolar and perivascular lymphoid tissue hyperplasia (14) was assessed by scoring mycoplasmal pneumonia lesions (0 to 6), while interstitial pneumonia lesions were scored (0 to 6) based on the severity of interstitial pneumonia, as described in a previous study (10). Mycoplasmal pneumonia lesions were confirmed by RT-PCR from lung lesions, also as described in a previous study (11).
Statistical analysis
Real-time PCR data were transformed to log10 values prior to statistical analysis evaluation. The Shapiro-Wilk test was then used to test the collected data for a normal distribution. One-way analysis of variance (ANOVA) was also used to examine whether there were statistically significant differences at each timepoint within the 6 groups. A 1-way ANOVA test result with such a statistical significance was further evaluated by conducting a post-hoc test for a pairwise comparison with Tukey’s adjustment. If the normality assumption was not met, the Kruskal-Wallis test was conducted. Results from the Kruskal-Wallis test that showed statistical significance were further evaluated with the Mann-Whitney U-test to include Tukey’s adjustment to compare the differences among the groups. Results were reported in P-value, with a value of P < 0.05 considered to be significant.
Results
Clinical observations
Pigs in the vaccinated/challenged groups (VacA-PM/Ch, VacB-PM/Ch, VacA-M+P/Ch, and VacB-M+P/Ch) had significantly lower (P < 0.05) mean respiratory scores at 0 to 77, 112, and 119 d post-challenge (dpc) than the unvaccinated/challenged group (UnVac/Ch). At 84 dpc, pigs in the VacA-PM/Ch group also had significantly lower (P < 0.05) mean respiratory scores than the UnVac/Ch group. At 91 dpc, the mean respiratory scores of pigs in both the VacA-PM/Ch and VacB-M+P/Ch groups were significantly lower (P < 0.05) than those of the UnVac/Ch group, while at 98 dpc, pigs in the VacA-PM/Ch, VacB-PM/Ch, and VacA-M+P/Ch groups had significantly lower (P < 0.05) mean respiratory scores than the UnVac/Ch group. At 105 dpc, pigs in the VacA-PM/Ch and VacA-M+P/Ch groups had a significantly lower (P < 0.05) respiratory score mean than those in the UnVac/Ch group. Pigs in the unvaccinated/unchallenged group (UnVac/UnCh) remained normal throughout the study (Figure 2).
Figure 2.
Clinical respiratory sign scores in the different groups: VacA-M+P/Ch (
); VacA-PM/Ch (
); VacB-M+P/Ch (
); VacB-PM/Ch (
); UnVac/Ch (■); and UnVac/UnCh (△). Different letters within a sampling point mean statistically significant differences (P < 0.05).
Growth performance
Average body weight (mean ± standard deviation) was calculated at 7 and 21 d old, which resulted in no observed statistical difference among the 6 groups. Average daily weight gain (ADWG) was also measured among the 6 groups. During the 21- to 56-day period, a statistical difference was not found, but this changed during the latter part of the study. The ADWG of pigs in the VacA-PM/Ch, VacB-PM/Ch, VacA-M+P/Ch, VacB-M+P/Ch, and UnVac/UnCh groups was significantly higher (P < 0.05) than that of the UnVac/Ch group during the 56- to 84-, 84- to 112, and 112- to 175-day periods A significantly higher overall growth rate (P < 0.05) was observed in all of these groups (VacA-PM/Ch, VacB-PM/Ch, VacA-M+P/Ch, VacB-M+P/Ch, and UnVac/UnCh) when compared to the UnVac/Ch group from 21- to 175-day-old (Table I).
Table I.
Average daily weight gain [(ADWG), mean ± standard deviation] in pigs from 6 groups.
| Period between days post-challenge | Age (days) | ADWG (grams/day/pig)* | |||||
|---|---|---|---|---|---|---|---|
|
| |||||||
| VacA-M+P/Ch | VacA-PM/Ch | VacB-M+P/Ch | VacB-PM/Ch | UnVac/Ch | UnVac/UnCh | ||
| −35 to 0 | 21 to 56 | 330.54 ± 23.81 | 332.85 ± 19.96 | 326.79 ± 29.54 | 330.71 ± 28.41 | 322.86 ± 22.34 | 326.07 ± 12.82 |
| 0 to 28 | 56 to 84 | 491.30 ± 38.95a | 494.42 ± 33.25a | 474.78 ± 41.13a | 478.13 ± 29.01a | 405.36 ± 19.19b | 500.45 ± 45.71a |
| 28 to 56 | 84 to 112 | 658.71 ± 37.23a | 658.26 ± 47.64a | 690.18 ± 61.47a | 664.06 ± 48.47a | 470.09 ± 36.27b | 698.21 ± 51.37a |
| 56 to 119 | 112 to 177 | 892.76 ± 43.05a | 894.35 ± 57.67a | 881.85 ± 28.77a | 885.52 ± 74.46a | 815.87 ± 07.05b | 909.13 ± 61.61a |
| −35 to 119 | 21 to 175 | 649.43 ± 20.02a | 651.10 ± 25.81a | 646.84 ± 14.19a | 645.09 ± 27.87a | 566.32 ± 09.32b | 663.96 ± 22.82a |
The live weight of each pig was measured at 21 d old [−35 days post-challenge (dpc)], 56 d old (0 dpc), 84 d old (28 dpc), 112 d old (56 dpc), and 175 d old (119 dpc; the time of final necropsy) among 6 groups.
Different superscript letters (a,b) within a sampling point mean statistically significant differences (P < 0.05).
Quantification of M. hyopneumoniae in nasal swabs
Prior to challenge, pigs in all groups were confirmed negative for M. hyopneumoniae (zero genomic copies) by PCR evaluation of nasal swabs. Nasal swabs were evaluated at multiple timepoints post-challenge. Pigs in the vaccinated-challenged groups (VacA-PM/Ch, VacB-PM/Ch, VacA-M+P/Ch, and VacB-M+P/Ch) had a significantly lower (P < 0.05) number of genomic copies of M. hyopneumoniae in their nasal swabs at 7, 14, 35, and 56 dpc than pigs in the unvaccinated challenged group (UnVac/Ch). At 14 and 35 dpc, pigs in the VacA-PM/Ch and VacA-M+P/Ch groups had a significantly lower (P < 0.05) number of genomic copies of M. hyopneumoniae in their nasal swabs than those in the VacB-PM/Ch and VacB-M+P/Ch groups (Figure 3A). All pigs in the unvaccinated/unchallenged group (UnVac/UnCh) remained free from M. hyopneumoniae (no genomic copies detected) throughout the nasal swab portion of this study.
Figure 3.
Quantification of M. hyopneumoniae (Mhp) DNA and PCV2 DNA. A — Mean values of the genomic copy numbers of M. hyopneumoniae DNA in the nasal swabs and B — Mean values of the genomic copy numbers of PCV2 DNA in the serum samples in the different groups: VacA-M+P/Ch (
); VacA-PM/Ch (
); VacB-M+P/Ch (
); VacB-PM/Ch (
); UnVac/Ch (■); and UnVac/UnCh (△). Different letters within a sampling point mean statistically significant differences (P < 0.05).
Quantification of PCV2d DNA in blood
Prior to challenge, pigs in all 6 groups were screened and found negative for PCV2 DNA. At 7 dpc, pigs in the VacA-PM/Ch and VacA-M+P/Ch groups had a significantly lower (P < 0.05) number of genomic copies of PCV2 in their blood than those in the VacB-PM/Ch, VacB-M+P/Ch, and UnVac/Ch groups. At 14, 35, and 56 dpc, pigs in the vaccinated/challenged groups (VacA-PM/Ch, VacB-PM/Ch, VacA-M+P/Ch, and VacB-M+P/Ch) had a significantly lower (P < 0.05) number of genomic copies of PCV2 in their blood than pigs in the unvaccinated/challenged group (UnVac/Ch). In the vaccinated/challenged groups, pigs in the VacA-PM/Ch and VacA-M+P/Ch groups had a significantly lower (P < 0.05) number of genomic copies of PCV2 in their blood at 14, 35, and 56 dpc than pigs in the VacB-PM/Ch and VacB-M+P/Ch groups (Figure 3B). No genomic copies of PCV2 were detected in any of the pigs in the unvaccinated/unchallenged group (UnVac/UnCh).
Enzyme-linked immunosorbent assay
Antibody responses against M. hyopneumoniae were assessed with ELISA. From −14 to 56 dpc, all vaccinated/challenged groups (VacA-PM/Ch, VacB-PM/Ch, VacA-M+P/Ch, and VacB-M+P/Ch) had a significantly higher (P < 0.05) M. hyopneumoniae ELISA S/P ratio than that of the unvaccinated/challenged group (UnVac/Ch). In the vaccinated/challenged groups, pigs in the VacA-M+P/Ch group had a significantly higher (P < 0.05) M. hyopneumoniae ELISA S/P ratio at −14 dpc than pigs in the VacB-PM/Ch and VacB-M+P/Ch groups. From 0 to 14 dpc, pigs in the VacA-M+P/Ch group had a significantly higher (P < 0.05) M. hyopneumoniae ELISA S/P ratio than pigs in the VacA-PM/Ch, VacB-PM/Ch, and VacB-M+P/Ch groups. Pigs in the VacA-PM/Ch group had a significantly higher (P < 0.05) M. hyopneumoniae ELISA S/P ratio at 35 and 56 dpc than pigs in the VacA-M+P/Ch, VacB-PM/Ch, and VacB-M+P/Ch groups (Figure 4A). No antibodies against M. hyopneumoniae were detected in any of the pigs from the unvaccinated/unchallenged group (UnVac/UnCh).
Figure 4.
Enzyme-linked immunosorbent assay. A — Mean values of the anti-M. hyopneumoniae (Mhp) antibody levels and B — Mean values of the anti-PCV2 antibody levels in the different groups: VacA-M+P/Ch (
); VacA-PM/Ch (
); VacB-M+P/Ch (
); VacB-PM/Ch (
); UnVac/Ch (■); and UnVac/UnCh (△). Different letters within a sampling point mean statistically significant differences (P < 0.05).
Antibody responses against PCV2 were also assessed with ELISA. From −14 to 56 dpc, pigs in the vaccinated/challenged groups (VacA-PM/Ch, VacB-PM/Ch, VacA-M+P/Ch, and VacB-M+P/Ch) had significantly higher (P < 0.05) PCV2 ELISA titers than pigs in the unvaccinated/challenged group (UnVac/Ch) (Figure 4B). No antibodies specific to PCV2 were detected in any of the pigs in the UnVac/UnCh group.
Enzyme-linked immunospot assay
T-cell response was determined as the number of M. hyopneumoniae-specific interferon-γ-secreting cells (IFN-γ-SCs) that was quantified in the peripheral blood mononuclear cells (PBMCs) of individual pigs. From −14 to 56 dpc, pigs in the vaccinated/challenged groups (VacA-PM/Ch, VacB-PM/Ch, VacA-M+P/Ch, and VacB-M+P/Ch) had a significantly higher (P < 0.05) number of M. hyopneumoniae-specific IFN-γ-SCs in their PBMCs than pigs in the UnVac/Ch group. In the vaccinated/challenged groups, pigs in the VacA-PM/Ch and VacA-M+P/Ch groups had a significantly higher (P < 0.05) number of M. hyopneumoniae-specific IFN-γ-SCs in their PBMCs at −14, 0, and 7 dpc than pigs in the VacB-PM/Ch and VacB-M+P/Ch groups.
At 14 dpc, pigs in the VacA-M+P/Ch group had a significantly higher (P < 0.05) number of M. hyopneumoniae-specific IFN-γ-SCs in their PBMCs than pigs in the VacB-PM/Ch group. Pigs in the VacA-PM/Ch group had a significantly higher (P < 0.05) number of M. hyopneumoniae-specific IFN-γ-SCs in their PBMCs at 35 dpc than pigs in the VacB-M+P/Ch group (Figure 5A). The mean numbers of M. hyopneumoniae-specific IFN-γ-SCs in the unvaccinated/unchallenged group (UnVac/UnCh) remained at basal levels (< 20 cells/106 PBMC) throughout the study.
Figure 5.
Numbers of interferon-γ-secreting cells (IFN-γ-SCs). A — Mean number of Mycoplasma hyopneumoniae (Mhp)-specific IFN-γ-SCs in peripheral blood mononuclear cells (PBMCs) and B — Mean number of PCV2-specific IFN-γ-SCs in PBMCs in the different groups: VacA-M+P/Ch (
); VacA-PM/Ch (
); VacB-M+P/Ch (
); VacB-PM/Ch (
); UnVac/Ch (■); and UnVac/UnCh (△). Different letters within a sampling point mean statistically significant differences (P < 0.05).
T-cell responses were also evaluated by comparing the number of PCV2-specific IFN-γ-SCs among groups. From −14 to 35 dpc, pigs in the vaccinated/challenged groups (VacA-PM/Ch, VacB-PM/Ch, VacA-M+P/Ch, and VacB-M+P/Ch) had a significantly higher (P < 0.05) number of PCV2d-specific IFN-γ-SCs in their PBMCs than pigs in the UnVac/Ch group. At 56 dpc, pigs in the VacA-PM/Ch group had a significantly higher (P < 0.05) number of PCV2d-specific IFN-γ-SCs in their PBMCs than those in the UnVac/Ch group.
In the vaccinated/challenged groups, pigs in the VacA-PM/Ch and VacA-M+P groups had a significantly higher (P < 0.05) number of PCV2d-specific IFN-γ-SCs in their PBMCs at −14 dpc than pigs in the VacB-M+P/Ch group. At 7, 14, and 35 dpc, pigs in the VacA-PM/Ch and VacA-M+P groups also displayed a significantly higher (P < 0.05) number of PCV2d-specific IFN-γ-SCs in their PBMCs than pigs in the VacB-PM/Ch and VacB-M+P/Ch groups (Figure 5B). The mean numbers of PCV2d-specific IFN-γ-SCs in the unvaccinated/unchallenged group (UnVac/UnCh) remained at basal levels (< 20 cells/106 PBMC) throughout the study.
Pathology
At 119 dpc, pigs in the vaccinated/challenged groups (VacA-PM/Ch, VacB-PM/Ch, VacA-M+P/Ch, and VacB-M+P/Ch) had significantly lower (P < 0.05) macroscopic lung lesions, microscopic lung lesions (mycoplasmal pneumonia and interstitial pneumonia), and lymphoid lesions scores than those of pigs in the unvaccinated/challenged group (UnVac/Ch). There were no macroscopic lung, microscopic lung, or lymphoid lesions in pigs in the unvaccinated/unchallenged group (UnVac/UnCh) (Table II).
Table II.
Pathology (mean ± standard deviation) in pigs from 6 groups at 119 d post-challenge (175 d old).
| Groups | ||||||
|---|---|---|---|---|---|---|
|
|
||||||
| Pathology | VacA-M+P/Ch | VacA-PM/Ch | VacB-M+P/Ch | VacB-PM/Ch | UnVac/Ch | UnVac/UnCh |
| Macroscopic lung lesions | 25.06 ± 2.02b | 25.88 ± 4.16b | 27.94 ± 3.94b | 26.19 ± 5.50b | 57.00 ± 2.67a | 4.75 ± 2.92c |
| Microscopic lung lesions | ||||||
| Mycoplasmal pneumonia | 1.44 ± 0.41b | 1.40 ± 0.28b | 1.43 ± 0.27b | 1.41 ± 0.46b | 2.98 ± 0.39a | 0.15 ± 0.14c |
| Interstitial pneumonia | 1.40 ± 0.30b | 1.30 ± 0.43b,c | 0.98 ± 0.44c | 1.34 ± 0.31b,c | 3.18 ± 0.45a | 0.23 ± 0.20d |
| Microscopic lymphoid lesions | 0.90 ± 0.24b | 0.88 ± 0.22b | 0.95 ± 0.45b | 0.85 ± 0.42b | 2.65 ± 0.52a | 0.18 ± 0.17c |
Different superscript letters (a,b,c) mean statistically significant differences (P < 0.05).
Discussion
This comparative trial demonstrates that vaccination against PCV2 and M. hyopneumoniae is efficacious in controlling these 2 pathogens, regardless of vaccine type, i.e., monovalent or bivalent. Economic losses related to PCV2 and M. hyopneumoniae are mainly associated with pig productivity. PCV2 vaccines are effective in reducing morbidity and mortality, while improving overall growth performance, even in pigs without clinical signs (15–19). Vaccination against M. hyopneumoniae reduces clinical signs and lung lesions while improving performance (20–24). Pigs are therefore often vaccinated against PCV2 and M. hyopneumoniae to improve growth performance even in the absence of clinical disease.
Evaluation of average daily weight gain (ADWG) is the main indicator of production performance and is therefore a critical parameter for comparing and selecting vaccines. All 4 vaccinated/challenged groups measured significantly higher growth performance than the unvaccinated/challenged group. A numerical difference in growth performance should be noted between the 2 VacA/challenged groups (VacA-PM/Ch and VacA-M+P/Ch) and the 2 VacB/challenged groups (VacB-PM/Ch and VacB-M+P/Ch). From 21 to 175 d old, ADWG was higher overall in the VacA/challenged groups than in the VacB/challenged groups, although the difference was too small to be considered statistically significant. This may be due to either the small number of animals per group or to the overly regulated experimental management conditions, both of which are different at a laboratory farm and in a commercial farm setting. Although not statistically significant, the observed numerical difference in ADWG may therefore still be clinically meaningful data for pig producers.
Although the exact protective mechanisms of M. hyopneumoniae vaccination are not yet fully understood, cell-mediated immunity may alleviate the development of mycoplasmal pneumonia (25). In this study, vaccination-generated interferon-γ-secreting cells (IFN-γ-SCs) within blood, which provides evidence that IFN-γ-SCs may play a role in protective cell-mediated immunity against M. hyopneumoniae (25). Pigs in the VacA/challenged groups (VacA-PM/Ch and VacA-M+P/Ch) generated significantly higher numbers of IFN-γ-SCs in their blood, while exhibiting less nasal shedding and mycoplasma pneumonia than those in the VacB/challenged groups (VacB-PM/Ch and VacB-M+P/Ch). It has been suggested that transmission through nasal secretions is a potential mode of horizontal spreading (26,27). As horizontal transmission is considered a main risk factor in chronically infected populations (26,27), minimizing nasal shedding of M. hyopneumoniae should be emphasized.
There are several potential reasons that the M. hyopneumoniae vaccines elicited different responses throughout the study. Each commercially available vaccine has unique antigen and adjuvant formulations (28). The vaccine strain used in the VacA/challenged groups (VacA-PM/Ch and VacA-M+P/Ch) also differed from the vaccine strains used in the VacB/challenged groups (VacB-PM/Ch and VacB-M+P/Ch) (29). Adjuvant formulation in particular is known to affect the immunogenicity and protective effect of inactivated whole-cell M. hyopneumoniae bacterins (30). The efficacy of an M. hyopneumoniae vaccine also differs depending on the challenge strain used to inoculate the animals (31). Each of these factors may have contributed to the outcome of this study. Further studies are needed to elucidate the difference in protective immune mechanisms and nasal shedding between M. hyopneumoniae vaccines.
For the M. hyopneumoniae vaccination, pigs in the monovalent groups (VacA-M+P/Ch and VacB-M+P/Ch) were vaccinated 2 calendar weeks before those in the bivalent groups (VacA-PM/Ch and VacB-PM/Ch). There was no significant difference, however, between vaccinating with either a monovalent vaccine at 1 wk of age or with a bivalent vaccine at 3 wk of age. It has been shown that vaccinating pigs at 7 d old with the same M. hyopneumoniae vaccine as used in this study was effective in reducing lung lesions, even in the presence of maternally derived antibodies (MDA) at a titer considerably higher than what is typically seen in the field (25). These antibodies therefore do not interfere with the onset of immunity in regard to the M. hyopneumoniae fraction of bivalent vaccines or with M. hyopneumoniae monovalent vaccines.
Vaccinating pigs for PCV2 has been proven effective for controlling PCVAD in pigs and inducing a strong protective immunity. In particular, as part of cell-mediated immune responses, IFN-γ-SCs production may be a key component in developing protective immunity against PCV2 infection (32). Failure to produce sufficient IFN-γ-SCs may result in lower titers of neutralizing antibodies and greater PCV2 viremia (33), which has been correlated to the severity of PCVAD (18,19,34). Increasing IFN-γ-SCs production therefore results in heightened protective immunity against PCV2 infection.
The present study demonstrates that the inactivated chimeric PCV1-2a vaccine in the VacA/challenged groups (VacA-PM/Ch and VacA-M+P/Ch) induced significantly higher numbers of PCV2d-specific IFN-γ-SCs than the inactivated subunit PCV2a vaccine based on the PCV2a capsid protein in the VacB/challenged groups (VacB-PM/Ch and VacB-M+P/Ch). The generation of higher amounts of IFN-γ-SCs by inactivated chimeric PCV1-2a vaccine results in less PCV2d viremia than that generated by the inactivated subunit PCV2a vaccine. Similarly, previous studies have resulted in inactivated chimeric PCV1-2a vaccine inducing higher numbers of PCV2b-specific IFN-γ-SCs and lower levels of PCV2b viremia (35). The different antigen and adjuvant formulations of the 2 PCV2 vaccines may account for these differences.
Most PCV2a-based monovalent vaccines now available provide cross-protection against PCV2d infection (36,37). The 2 PCV2a-based monovalent and bivalent vaccines used in this study also provide cross-protection against PCV2d. This was a critical parameter as PCV2d is the predominant genotype in Asia, including Korea. According to the Korea Animal Health Products Association (http://www.kahpa.or.kr), almost all piglets farrowed in 2018 were administered some form of a PCV2 vaccine.
Although antigen interference is always a concern within a bivalent vaccine, the present study showed similar generation of protective immunity, such as neutralization antibodies and IFN-γ-SCs, and reduction of PCV2d viremia regardless of whether the vaccine was a bivalent or a PCV2 monovalent. In addition, no significant difference was observed between bivalent and mycoplasmal monovalent in the generation of IFN-γ-SCs and reduction of nasal shedding of M. hyopneumoniae, which suggests that there was no antigen interference.
Porcine circovirus associated disease (PCVAD) and enzootic pneumonia are considered the 2 most prominent and costly diseases in pigs at the present time. Certain factors should be considered when interpreting the results of this study. The results obtained from the experimental conditions described in this study are not indicative of field conditions. The immunological background (i.e., maternally derived antibodies), timing of infection, and other factors, such as management, production system, and facility, including ventilation, can greatly influence the efficacy of a vaccination program. Swine practitioners and producers therefore need to consider variables when applying the results of this study to commercial pig farms.
Acknowledgments
This study was supported by contract research funds from the Research Institute for Veterinary Science (RIVS) at the College of Veterinary Medicine and by the Brain Korea 21 Plus Program (Grant no. 5260-20150100) for Creative Veterinary Science Research in the Republic of Korea.
References
- 1.Chae C. A review of porcine circovirus 2-associated syndromes and diseases. Vet J. 2005;169:326–336. doi: 10.1016/j.tvjl.2004.01.012. [DOI] [PubMed] [Google Scholar]
- 2.Davies B, Wang X, Dvorak CMT, Marthaler D, Murtaugh MP. Diagnostic phylogenetics reveals a new porcine circovirus 2 cluster. Virus Res. 2016;217:32–37. doi: 10.1016/j.virusres.2016.02.010. [DOI] [PubMed] [Google Scholar]
- 3.Franzo G, Segalés J. Porcine circovirus 2 (PCV-2) genotype update and proposal of a new genotyping methodology. PLoS ONE. 2018;13:e0208585. doi: 10.1371/journal.pone.0208585. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Seo HW, Park C, Kang I, et al. Genetic and antigenic characterization of a newly emerging porcine circovirus type 2b mutant first isolated in cases of vaccine failure in Korea. Arch Virol. 2014;159:3107–3111. doi: 10.1007/s00705-014-2164-6. [DOI] [PubMed] [Google Scholar]
- 5.Thacker EL, Minion C. Mycoplasmosis. In: Zimmerman JJ, Karriker LA, Ramirez A, Schwartz KJ, Stevenson GW, editors. Diseases of Swine. 10th ed. Ames, Iowa: Wiley-Blackwell; 2012. pp. 779–797. [Google Scholar]
- 6.Razin S, Yogev D, Naot Y. Molecular biology and pathogenicity of mycoplasmas. Microbiol Mol Biol Rev. 1998;62:1094–1156. doi: 10.1128/mmbr.62.4.1094-1156.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.Van Reeth K, Nauwynck H, Pensaert M. A potential role for tumour necrosis factor-alpha in synergy between porcine respiratory coronavirus and bacterial lipopolysaccharide in the induction of respiratory disease in pigs. J Med Microbiol. 2000;49:613–620. doi: 10.1099/0022-1317-49-7-613. [DOI] [PubMed] [Google Scholar]
- 8.Marchioro SB, Sacristan RDP, Michiels A, et al. Immune responses of a chimeric protein vaccine containing Mycoplasma hyopneumoniae antigens and LTB against experimental M. hyopneumoniae infection in pigs. Vaccine. 2014;32:4689–4694. doi: 10.1016/j.vaccine.2014.05.072. [DOI] [PubMed] [Google Scholar]
- 9.Beaver BV, Reed W, Leary S, et al. 2000 Report of the AVMA panel on euthanasia. J Am Vet Med Assoc. 2001;218:669–696. doi: 10.2460/javma.2001.218.669. [DOI] [PubMed] [Google Scholar]
- 10.Halbur PG, Paul PS, Frey ML, et al. Comparison of the pathogenicity of two US porcine reproductive and respiratory syndrome virus isolates with that of the Lelystad virus. Vet Pathol. 1995;32:648–660. doi: 10.1177/030098589503200606. [DOI] [PubMed] [Google Scholar]
- 11.Dubosson CR, Conzelmann C, Miserez R, et al. Development of two real-time PCR assays for the detection of Mycoplasma hyopneumoniae in clinical samples. Vet Microbiol. 2004;102:55–65. doi: 10.1016/j.vetmic.2004.05.007. [DOI] [PubMed] [Google Scholar]
- 12.Jeong J, Park C, Choi K, Chae C. Comparison of three commercial one-dose porcine circovirus type 2 (PCV2) vaccines in a herd with concurrent circulation of PCV2b and mutant PCV2b. Vet Microbiol. 2015;177:43–52. doi: 10.1016/j.vetmic.2015.02.027. [DOI] [PubMed] [Google Scholar]
- 13.Jeong J, Kang I, Kim S, Park KH, Park C, Chae C. Comparison of 3 vaccination strategies against porcine reproductive and respiratory syndrome virus, Mycoplasma hyopneumoniae, and porcine circovirus type 2 on a 3 pathogen challenge model. Can J Vet Res. 2018;82:39–47. [PMC free article] [PubMed] [Google Scholar]
- 14.Thacker EL, Halbur PG, Ross RF, Thanawongnuwech R, Thacker BJ. Mycoplasma hyopneumoniae potentiation of porcine reproductive and respiratory syndrome virus-induced pneumonia. J Clin Microbiol. 1999;37:620–627. doi: 10.1128/jcm.37.3.620-627.1999. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15.Fachinger V, Bischoff R, Jedidia SB, Saalmüller A, Elbers K. The effect of vaccination against porcine circovirus type 2 in pigs suffering from porcine respiratory disease complex. Vaccine. 2008;26:1488–1499. doi: 10.1016/j.vaccine.2007.11.053. [DOI] [PubMed] [Google Scholar]
- 16.Horlen KP, Dritz SS, Nietfeld JC, et al. A field evaluation of mortality rate and growth performance in pigs vaccinated against porcine circovirus type 2. J Am Vet Med Assoc. 2008;232:906–912. doi: 10.2460/javma.232.6.906. [DOI] [PubMed] [Google Scholar]
- 17.Kixmöller M, Ritzmann M, Eddicks M, Saalmüller A, Elbers K, Fachinger V. Reduction of PMWS-associated clinical signs and co-infections by vaccination against PCV2. Vaccine. 2008;26:3443–3451. doi: 10.1016/j.vaccine.2008.04.032. [DOI] [PubMed] [Google Scholar]
- 18.Martelli P, Ferrari L, Morganti M, et al. One dose of a porcine circovirus 2 subunit vaccine induces humoral and cell-mediated immunity and protects against porcine circovirus-associated disease under field conditions. Vet Microbiol. 2011;149:339–351. doi: 10.1016/j.vetmic.2010.12.008. [DOI] [PubMed] [Google Scholar]
- 19.Seo HW, Han K, Oh Y, Park C, Chae C. Efficacy of a reformulated inactivated chimeric PCV1-2 vaccine based on clinical, virological, pathological and immunological examination under field conditions. Vaccine. 2012;30:6671–6677. doi: 10.1016/j.vaccine.2012.08.065. [DOI] [PubMed] [Google Scholar]
- 20.Del Pozo Sacristán R, Sierens A, Marchioro SB, et al. Efficacy of early Mycoplasma hyopneumoniae vaccination against mixed respiratory disease in older fattening pigs. Vet Rec. 2014;174:197. doi: 10.1136/vr.101597. [DOI] [PubMed] [Google Scholar]
- 21.Jensen CS, Ersbøll AK, Nielsen JP. A meta-analysis comparing the effect of vaccines against Mycoplasma hyopneumoniae on daily weight gain in pigs. Prev Vet Med. 2002;54:265–278. doi: 10.1016/s0167-5877(02)00005-3. [DOI] [PubMed] [Google Scholar]
- 22.Maes D, Deluyker H, Verdonck M, et al. The effect of vaccination against Mycoplasma hyopneumoniae in pig herds with a continuous production system. Zentralbl Veterinarmed B. 1998;45:495–505. doi: 10.1111/j.1439-0450.1998.tb00820.x. [DOI] [PubMed] [Google Scholar]
- 23.Maes D, Deluyker H, Verdonck M, et al. Effect of vaccination against Mycoplasma hyopneumoniae in pig herds with an all-in/all-out production system. Vaccine. 1999;17:1024–1034. doi: 10.1016/s0264-410x(98)00254-0. [DOI] [PubMed] [Google Scholar]
- 24.Wilson S, Van Brussel L, Saunders G, et al. Vaccination of piglets at 1 week of age with an inactivated Mycoplasma hyopneumoniae vaccine reduces lung lesions and improves average daily gain in body weight. Vaccine. 2012;30:7625–7629. doi: 10.1016/j.vaccine.2012.10.028. [DOI] [PubMed] [Google Scholar]
- 25.Thacker EL, Thacker BJ, Kuhn M, Hawkins PA, Waters WR. Evaluation of local and systemic immune responses induced by intramuscular injection of a Mycoplasma hyopneumoniae bacterin to pigs. Am J Vet Res. 2000;61:1384–1389. doi: 10.2460/ajvr.2000.61.1384. [DOI] [PubMed] [Google Scholar]
- 26.Clark LK, Armstrong CH, Scheidt AB, Van Alstine WG. The effect of Mycoplasma hyopneumoniae infection on growth in pigs with or without environmental constraints. Swine Health Prod. 1993;6:10–14. [Google Scholar]
- 27.Fano E, Pijoan C, Dee S. Dynamics and persistence of Mycoplasma hyopneumoniae infection in pigs. Can J Vet Res. 2005;69:223–228. [PMC free article] [PubMed] [Google Scholar]
- 28.Maes D, Sibila M, Kuhnert P, Segalés J, Haesebrouck F, Pieters M. Update on Mycoplasma hyopneumoniae infection in pigs: Knowledge gaps for improved disease control. Transbound Emerg Dis. 2018;65( Suppl 1):110–124. doi: 10.1111/tbed.12677. [DOI] [PubMed] [Google Scholar]
- 29.Tao Y, Shu J, Chen J, Wu Y, He Y. A concise review of vaccines against Mycoplasma hyopneumoniae. Res Vet Sci. 2019;123:144–152. doi: 10.1016/j.rvsc.2019.01.007. [DOI] [PubMed] [Google Scholar]
- 30.Galliher-Beckley A, Pappan LK, Madera R, et al. Characterization of a novel oil-in-water emulsion adjuvant for swine influenza virus and Mycoplasma hyopneumoniae vaccines. Vaccine. 2015;33:2903–2908. doi: 10.1016/j.vaccine.2015.04.065. [DOI] [PubMed] [Google Scholar]
- 31.Villarreal I, Maes D, Vranckx K, Calus D, Pasmans F, Haesebrouck F. Effect of vaccination of pigs against experimental infection with high and low virulence Mycoplasma hyopneumoniae strains. Vaccine. 2011;29:1731–1735. doi: 10.1016/j.vaccine.2011.01.002. [DOI] [PubMed] [Google Scholar]
- 32.Fort M, Fernandes LT, Nofrarias M, et al. Development of cell-mediated immunity to porcine circovirus type 2 (PCV2) in caesarean-derived, colostrum-deprived piglets. Vet Immunol Immunopathol. 2009;129:101–107. doi: 10.1016/j.vetimm.2008.12.024. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 33.Meerts P, Van Gucht S, Cox E, Vandebosch A, Nauwynck HJ. Correlation between type of adaptive immune response against porcine circovirus type 2 and level of virus replication. Viral Immunol. 2005;18:333–341. doi: 10.1089/vim.2005.18.333. [DOI] [PubMed] [Google Scholar]
- 34.Fort M, Sibila M, Allepuz A, Mateu E, Roerink F, Segalés J. Porcine circovirus type 2 (PCV2) vaccination of conventional pigs prevents viremia against PCV2 isolates of different genotypes and geographic origins. Vaccine. 2009;26:1063–1071. doi: 10.1016/j.vaccine.2007.12.019. [DOI] [PubMed] [Google Scholar]
- 35.Seo HW, Han K, Park C, Chae C. Clinical, virological, immunological and pathological evaluation of four porcine circovirus type 2 vaccines. Vet J. 2014;200:65–70. doi: 10.1016/j.tvjl.2014.02.002. [DOI] [PubMed] [Google Scholar]
- 36.Opriessnig T, Gerber PF, Xiao C-T, Halbur PG, Matzinger SR, Meng X-J. Commercial PCV2a-based vaccines are effective in protecting naturally PCV2b-infected finisher pigs against experimental challenge with a 2012 mutant PCV2. Vaccine. 2014;32:4342–4348. doi: 10.1016/j.vaccine.2014.06.004. [DOI] [PubMed] [Google Scholar]
- 37.Park KH, Oh T, Yang S, Cho H, Kang I, Chae C. Evaluation of a porcine circovirus type 2a (PCV2a) vaccine efficacy against experimental PCV2a, PCV2b, and PCV2d challenge. Vet Microbiol. 2019;231:87–92. doi: 10.1016/j.vetmic.2019.03.002. [DOI] [PubMed] [Google Scholar]