Abstract
The objective of this study was to compare clinical, microbiologic, immunologic, and pathologic parameters in pigs each concurrently administered porcine reproductive and respiratory syndrome virus (PRRSV), Mycoplasma hyopneumoniae, and porcine circovirus type 2 (PCV2) vaccine from 1 of 2 commercial sources at 21 days of age and challenged with field strains of each of the 3 pathogens. Pigs were challenged with PRRSV and M. hyopneumoniae at 42 days of age (−14 days post-challenge, dpc) followed by a challenge with PCV2 at 56 days of age (0 dpc). Significant differences were observed between vaccinated challenged and unvaccinated challenged groups in clinical (average daily gain and clinical signs), microbiologic (viremia and nasal shedding), immunologic (antibodies and interferon-γ secreting cells), and pathologic (lesions) outcomes. Significant differences were observed among the 3 vaccinated challenged groups in microbiologic (nasal shedding of M. hyopneumoniae and viremia of PCV2) and immunologic (M. hyopneumoniae- and PCV2-specific interferon-γ secreting cells) outcomes. The vaccination regimen for PRRSV vaccine, M. hyopneumoniae vaccine, and PCV2 vaccine is efficacious for controlling triple challenge with PRRSV, M. hyopneumoniae, and PCV2 from weaning to finishing period.
Résumé
L’objectif de la présente étude était de comparer les paramètres cliniques, microbiologiques, immunologiques et pathologiques chez des porcs qui ont chacun été vaccinés de façon concomitante à 21 jours d’âge contre le virus du syndrome reproducteur et respiratoire porcin (VSRRP), Mycoplasma hyopneumoniae, et le circovirus porcin de type 2 (CVP2) provenant d’une de deux sources commerciales et soumis à une infection défi avec des souches de champs de chacun des trois agents pathogènes. Les porcs ont été infectés avec le VSRRP et M. hyopneumoniae à 42 jours d’âge (−14 jours post-infection, jpi) suivi d’une infection par le CVP2 à 56 jours d’âge (0 jpi). Des différences significatives ont été observées entre les groupes vaccinés et infectés et les groupes non-vaccinés infectés pour ce qui est résultats cliniques (gain moyen quotidien et signes cliniques), microbiologiques (virémie et excrétion nasale), immunologiques (anticorps et cellules secrétant de l’interféron-γ), et pathologiques (lésions). Des différences significatives ont été observées entre les trois groupes d’animaux vaccinés et infectés pour ce qui est des résultats microbiologiques (excrétion nasale de M. hyopneumoniae et virémie de CVP2) et immunologiques (cellules secrétant de l’interféron-γ spécifiques à M. hyopneumoniae et CVP2). Le protocole de vaccination pour le vaccin VSRRP, le vaccin M. hyopneumoniae, et le vaccin CVP2 est efficace pour la maitrise d’une infection triple avec le VSRRP, M. hyopneumoniae, et CVP2 à partir du sevrage jusqu’à la période de finition.
(Traduit par Docteur Serge Messier)
Introduction
Porcine respiratory disease complex (PRDC) occurs most commonly in pigs at 16 to 20 wk of age. Affected pigs exhibit cough and fever, and stop eating. These pigs have a poor growth performance (1,2). Porcine respiratory disease complex is the result of a combination of multiple infectious pathogens, environmental stressors, and production system challenges (1,2). Porcine reproductive and respiratory syndrome virus (PRRSV) and Mycoplasma hyopneumoniae are generally involved in PRDC (2). In addition, porcine circovirus type 2 (PCV2) can play a role with these 2 pathogens or independently of any other infectious pathogen in causing PRDC (3). It is now clear that PRRSV, M. hyopneumoniae, and PCV2 are recognized as major contributors to PRDC (1).
During the early 2000s, porcine circovirus-associated disease (PCVAD) caused significant economic losses on pig production throughout Asia (4,5). After the introduction of PCV2 vaccine around 2007 to 2008, PCVAD has been well controlled (6). However, even if the PCV2 vaccine is widely used, the PRDC continues to develop and cause severe economic losses in most Asian pig farms. Currently, PRDC has become one of the most important economic diseases with its rapidly increasing prevalence in Asian pig industry (1).
Porcine respiratory disease complex presents a substantial challenge to both veterinarians and producers today. Implementing strategic vaccination is essential in controlling PRDC. In the field, swine producers usually administer single-doses of PRRSV, M. hyopneumoniae, and PCV2 vaccines to control PRDC. However, no one has reported a comparison study of commercial single-dose of PRRSV, M. hyopneumoniae, and PCV2 vaccines that mirror field conditions. The objective of this study was to compare, through clinical, microbiologic, immunologic, and pathologic parameters, the efficacy of 3 vaccines from 2 commercial sources administered in 3 different ways in pigs that were challenged with a field strain of each of the 3 pathogens.
Materials and methods
Commercial vaccines
Two inactivated M. hyopneumoniae bacterins were used in this study: RespiSure-One (Lot No. 111914; Zoetis, Parsippany, New Jersey, USA) and Ingelvac MycoFLEX (Lot No. 2730377A; Boehringer Ingelheim Vetmedica, St. Joseph, Missouri, USA). Two modified live PRRSV vaccines were used in this study: Fostera PRRS (Lot No. 114917; Zoetis) and Ingelvac PRRS MLV (Lot No. 2451017B; Boehringer Ingelheim Vetmedica). Two inactivated PCV2 vaccines were used in this study: Fostera PCV MetaStim (Lot No. 1501509B; Zoetis) and Ingelvac CircoFLEX (Lot No. 3090944A; Boehringer Ingelheim Vetmedica). One bivalent PCV2-M. hyopneumoniae vaccine (Fostera PCV MH, Lot No. 106468B; Zoetis) was also used in this study. All vaccines used in this study were administered according to the manufacturer’s label claims with regards to timing and route of injection (intramuscularly).
Inocula
The PRRSV strain SNUVR090851 (type 2 genotype, lineage 1, GenBank JN315685), M. hyopneumoniae strain SNU98703, and PCV2b strain SNUVR000463 (GenBank KF871068) were used as inocula. Co-infection with PCV2 strain SNUVR000463 and M. hyopneumoniae strain SNU98703 induced severe pneumonia in lungs and lymphoid depletion in the lymph node in infected pigs (7). Similarly, co-infection with the identical PCV2 strain SNUVR000463 and PRRSV strain SNUVR090851 also induced similar symptoms as did the previous co-infection (8).
Animals
A total of 84 colostrum-fed, cross-bred, conventional piglets were purchased at 18 d of age from a PRRSV- and M. hyopneumoniae-free commercial farm based on serological testing of the breeding herd, and long term clinical and slaughter history. At 21 d of age, pigs were seronegative for PRRSV, M. hyopneumoniae, and PCV2. Similarly, negative results were also obtained from sera samples and nasal swabs for PCV2 and PRRSV, and for M. hyopneumoniae, respectively, when tested by real-time polymerase chain reaction (RT-PCR) (9–11).
Experimental design
A total of 84 pigs were randomly divided into 5 groups using random number generation function (Excel; Microsoft Corporation, Redmond, Washington, USA; Table I). The groups were Zoetis-VacA/Ch, Zoetis-VacB/Ch, BI-Vac/Ch, and UnVac/Ch (n = 18 for all 4 groups). The pigs in groups were randomly assigned into 8 out of 9 rooms. Each room had 12 pens and each pig was housed individually in a pen. In each of the 8 rooms, allocation of pens to treatment was in accordance with a randomized complete block design with 1-way treatment structure. Blocking was based on pen location. A block comprised 4 pens located near each other. The experimental unit for the treatment was the animal. Within each block, one pen was randomly assigned to each treatment group (Zoetis-VacA/Ch, Zoetis-VacB/Ch, BI-Vac/Ch, and UnVac/Ch). The pigs in UnVac/UnCh (n = 12) group were randomly placed into 12 pens in the one remaining room.
Table I.
Experimental design with vaccination and challenge statuses for porcine reproductive and respiratory syndrome virus (PRRSV), Mycoplasma hyopneumoniae (Mhp), and porcine circovirus type 2 (PCV2) at different days post-challenge (dpc).
| Vaccination (dpc) | Challenge (dpc) | Necropsy (dpc) | |||
|---|---|---|---|---|---|
|
|
|
|
|||
| Group | −49 | −35 | −14 | 0 | 119 |
| Age (days) | 7 | 21 | 42 | 56 | 175 |
| Zoetis-VacA/Ch | RespiSure-One | Fostera PCV MetaStim Fostera PRRS |
PRRSV/Mhp | PCV2 | 18 pigs |
| Zoetis-VacB/Ch | None | Fostera PCV MH Fostera PRRS |
PRRSV/Mhp | PCV2 | 18 pigs |
| BI-Vac/Ch | None | CircoFLEX-MycoFLEX Ingelvac PRRS MLV |
PRRSV/Mhp | PCV2 | 18 pigs |
| UnVac/Ch | None | None | PRRSV/Mhp | PCV2 | 18 pigs |
| UnVac/UnCh | None | None | None | None | 12 pigs |
For every vaccine mentioned, each dose contained 2.0 mL, and was administered into separate anatomical sites on the neck to prevent the vaccines from forming a mixture and relevant interference with the effects of vaccines. At −49 d post-challenge (dpc, 7 d of age), the pigs in Zoetis-VacA/Ch group were administered one dose of RespiSure-One on the left side of the neck. At −35 dpc (21 d of age), the pigs in Zoetis-VacA/Ch group were concurrently administered one dose of Fostera PCV MetaStim on the left side of the neck, as well as a dose of Fostera PRRS on the right side of the neck. The pigs in Zoetis-VacB/Ch group were concurrently administered one dose of each of Fostera PCV MH on the left side of the neck and Fostera PRRS on the right side of the neck. The pigs in BI-Vac/Ch group were concurrently administered one dose of Ingelvac CircoFLEX-MycoFLEX on the left side of the neck and one dose of Ingelvac PRRS MLV on right side of the neck. For Ingelvac CircoFLEX-MycoFLEX, separately contained vaccines were mixed prior to administration. The pigs in UnVac/Ch and UnVac/UnCh groups were administered one dose of phosphate-buffered saline (PBS, 0.01M, pH 7.4).
At −14 dpc (42 d of age), the pigs in Zoetis-VacA/Ch, Zoetis-VacB/Ch, BI-Vac/Ch, and UnVac/Ch groups were inoculated with PRRSV and M. hyopneumoniae. For inoculation, a 5 h interval was chosen after PRRSV inoculation before inoculating with M. hyopneumoniae to avoid mixture of the 2 pathogens which may decrease infectivity. Pigs were intranasally administered a 3 mL inoculation of PRRSV containing 1.2 × 105 50% tissue culture infective dose (TCID50)/mL. Five hours after PRRSV inoculation, pigs were anesthetized with a mixture of 2.2 mg/kg BW xylazine hydrochloride (Rumpon; Bayer Korea, Seoul, Korea) and 2.2 mg/kg BW tiletamine hydrochloride and 2.2 mg/kg BW zolazepam hydrochloride (Zoletil 50; Virbac Korea, Seoul, Korea) by intramuscular injection, and were inoculated intratracheally with 7 mL of M. hyopneumoniae culture medium containing 107 color changing units (CCU)/mL as previously described (12,13). At 0 dpc (56 d of age), pigs in Zoetis-VacA/Ch, Zoetis-VacB/Ch, BI-Vac/Ch, and UnVac/Ch groups were intranasally administered a 3 mL inoculation of PCV2 containing 1.2 × 105 TCID50/mL. The pigs in UnVac/UnCh group served as the negative control group.
Blood and nasal swabs were collected at −35, −28, −14, −7, 0, 7, 14, 56, and 119 dpc. All pigs were sedated using an intravenous injection of sodium pentobarbital and then euthanized by electrocution at 119 dpc as previously described (14). Tissues were collected from each pig at necropsy. Tissues were fixed for 24 h in 10% neutral buffered formalin, routinely processed, and embedded in paraffin. All of the methods were previously approved by the Seoul National University Institutional Animal Care and Use, and Ethics Committee.
Clinical observation
The pigs were monitored daily for clinical signs and scored weekly using scores ranging from 0 (normal) to 6 (severe dyspnea and abdominal breathing) (15). 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.
The live weight of each pig was measured at −35 dpc (21 d of age), 0 dpc (56 d of age), 28 dpc (84 d of age), 56 dpc (112 d of age), 84 dpc (140 d of age), and 119 dpc (175 d of age). The average daily gain (ADG; grams/pig/day) was analyzed over 5 time periods: i) between −35 and 0 dpc, ii) between 0 and 28 dpc, iii) between 28 and 56 dpc, iv) between 56 and 84 dpc, and v) between 84 and 119 dpc. The ADG 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 PRRSV RNA in blood
The RNA was extracted from serum samples to quantify PRRSV genomic cDNA copy numbers, as previously described (10). Real-time PCR for PRRSV was done to quantify PRRSV genomic cDNA copy (10). Real-time PCR for the vaccine viruses was also done to quantify PRRSV genomic cDNA copy (16,17).
Quantification of M. hyopneumoniae DNA in nasal swabs
The DNA was extracted from nasal swabs using a commercial kit (QIAamp DNA Mini Kit; QIAGEN, Valencia, California, USA) to quantify the M. hyopneumoniae genomic DNA copy numbers by RT-PCR (11).
Quantification of PCV2 DNA in blood
The DNA was extracted from serum samples using a commercial kit (QIAamp DNA Mini Kit; QIAGEN) to quantify PCV2 genomic DNA copy numbers by RT-PCR (9).
Serology
The serum samples were tested for antibodies to PRRSV (IDEXX PRRS X3 Ab test; IDEXX Laboratories, Westbrook, Maine, USA), M. hyopneumoniae (M. hyo. Ab test; IDEXX Laboratories), and PCV2 (PCV2 Ab Mono Blocking; Synbiotics, Lyon, France). Serum samples were considered positive for PRRSV and M. hyopneumoniae antibody if the S/P ratio was equal to or more than 0.4. Serum samples were considered positive for PCV2 IgG antibody if the reciprocal ELISA titer was greater than 150 according to the manufacturer’s instructions.
Enzyme-linked immunospot assay
The numbers of PRRSV-specific interferon-γ secreting cells (IFNγ-SC) were determined in peripheral blood mononuclear cells (PBMC) using the challenging PRRSV strain as previously described (17–19). The numbers of PCV2- and M. hyopneumoniae-specific IFNγ-SC were determined in PBMC using the challenging PCV2 and M. hyopneumoniae strain, respectively, as previously described (19,20).
Pathology
The total extent of gross lung lesions was estimated and calculated as previously described (21). The frequency distribution of the lung lesion scores for each lung lobe was calculated by treatment. The percentage of total lung with lesions was calculated using the following formula: 100 × [(0.10 × left cranial) + (0.1 × left middle) + (0.25 × left caudal) + (0.10 × right cranial) + (0.10 × right middle) + (0.25 × right caudal) + (0.10 × accessory)].
Lung and lymphoid tissue sections were blindly examined by 2 veterinary pathologists (Jeong and Chae). Mycoplasmal pneumonia lesions were scored (0 to 4) based on the severity of peribronchiolar and perivascular lymphoid tissue hyperplasia (22). Interstitial pneumonia lesions were scored (0 to 4) based on the severity of interstitial pneumonia as previously described (15). The PCV2-associated lymphoid lesions were scored (0 to 3) based on the severity of lymphoid depletion and granulomatous inflammation (22).
Immunohistochemistry and in situ hybridization
Immunohistochemistry (IHC) for PRRSV and PCV2 was done as previously described (8,23). In situ hybridization (ISH) for M. hyopneumoniae was done as previously described (24). For the morphometric analyses of ISH and IHC, 3 sections were cut from each of 3 blocks of tissue from lung and lymph node of each pig. The slides were analyzed using computer software (NIH Image J 1.51r Program, National Institute of Health, Bethesda, Maryland, USA) to obtain the quantitative data. For the analysis of PRRSV and PCV2, 10 fields were randomly selected, and the number of positive cells per unit area (0.95 mm2) was determined as previously described (25,26). For the analysis of M. hyopneumoniae, 10 fields were randomly selected and slides were scored ranging from 0 (no signal detectable) to 3 (intense labeling on the surface of several airway) as previously described (22). The mean values were also calculated.
Statistical analyses
A generalized linear mixed model for repeated measures was used to analyze the ADG, ELISA, PRRSV RNA quantification, M. hyopneumoniae DNA quantification, and PCV2 DNA quantification. Using a generalized linear mixed model for repeated measures or proportion test was used for the dichotomized endpoints (using a clinical cutoff to define normal/abnormal status of each animal): clinical score PRRSV induced lung microscopic lesion score, mycoplasmal lung microscopic lesion scores, PCV2-associated lymphoid microscopic lesions core, IHC of PRRSV and PCV2, and ISH of M. hyopneumoniae. The fixed effects of the generalized linear mixed model included treatment, time point, and their interaction. The random effects of the model included room, block within room, and animal. If only a single time point was measured, the time point related fixed effects and the animal term was excluded from the model. A value of P < 0.05 was considered to be significant.
Results
Clinical observation
Pigs in the Zoetis-VacA/Ch, Zoetis-VacB/Ch, and BI-Vac/Ch groups remained normal throughout the study, as measured by their respiratory scores, whereas moderate to severe respiratory signs were observed in UnVac/Ch group. The mean respiratory scores were significantly higher (P < 0.05) in pigs from UnVac/Ch group compared to pigs from Zoetis-VacA/Ch, Zoetis-VacB/Ch, BI-Vac/Ch, and UnVac/UnCh groups at −7 to 119 dpc (Figure 1). The overall mortality rate was significantly lower (P < 0.05) in pigs from Zoetis-VacA/Ch (0.06%, 1/18), Zoetis-VacB/Ch (0.06%, 1/18), BI-Vac/Ch (0.06%, 1/18), and UnVac/UnCh (0%) groups compared to pigs from UnVac/Ch (0.22%, 4/18) group. Diagnostic results indicated that mortality was primarily related to severe pneumonia and granulomatous lymphadenitis.
Figure 1.
Clinical respiratory sign scores in the different groups: Zoetis-VacA/Ch (
), Zoetis-VacB/Ch (
), BI-Vac/Ch (
), UnVac/Ch (
), and UnVac/UnCh (⋄). Different letters within a sampling point mean statistically significant differences (P < 0.05).
The overall ADG (from 3 to 25 wk of age) in pigs from Zoetis-VacA/Ch, Zoetis-VacB/Ch, BI-Vac/Ch, and UnVac/UnCh groups was significantly higher (P < 0.05) compared to UnVac/Ch group from −7 to 119 dpc. There was no significant difference in the ADG among Zoetis-VacA/Ch, Zoetis-VacB/Ch, and BI-Vac/Ch groups (Table II).
Table II.
Average daily gain (ADG, mean ± standard deviation) in pigs from different groups.
| Period between days post-challenge | Age (days) | ADG (grams/day/pig) | ||||
|---|---|---|---|---|---|---|
|
| ||||||
| Zoetis-VacA/Ch | Zoetis-VacB/Ch | BI-Vac/Ch | UnVac/Ch | UnVac/UnCh | ||
| −35 to 0 | 21 to 56 | 376.19 ± 27.50 | 375.71 ± 31.90 | 373.65 ± 33.20 | 380.63 ± 35.82 | 381.90 ± 36.11 |
| 0 to 28 | 56 to 84 | 626.19 ± 44.22a | 628.57 ± 50.68a | 623.81 ± 43.40a | 455.13 ± 49.95b | 671.13 ± 39.57a |
| 28 to 56 | 84 to 112 | 685.08 ± 36.47a | 677.73 ± 58.32a | 685.71 ± 78.18a | 584.89 ± 35.83b | 694.94 ± 38.45a |
| 56 to 84 | 112 to 140 | 732.35 ± 50.32a | 735.50 ± 46.30a | 710.50 ± 59.42a | 550.60 ± 81.10b | 715.48 ± 42.67a |
| 84 to 119 | 140 to 175 | 732.94 ± 40.03a | 720.17 ± 35.22a | 727.14 ± 37.06a | 655.24 ± 87.87b | 724.29 ± 28.32a |
| −35 to 119 | 21 to 175 | 625.29 ± 15.53a | 622.08 ± 17.31a | 620.30 ± 14.15a | 531.49 ± 22.03b | 629.87 ± 14.31a |
Different letters within a sampling point mean statistically significant differences (P < 0.05).
Quantification of PRRSV RNA in blood
At the time of challenge, no genomic copies of PRRSV were detected in any of the serum samples from any of the 5 groups. Pigs in the Zoetis-VacA/Ch, Zoetis-VacB/Ch, and BI-Vac/Ch groups had a significantly lower (P < 0.05) number of genomic copies of PRRSV in their sera than pigs in UnVac/Ch group from −7 to 56 dpc. No genomic copies of PRRSV were detected in any of the serum samples from UnVac/UnCh group throughout the study (Figure 2A).
Figure 2.
Quantification of porcine reproductive and respiratory syndrome virus (PRRSV) RNA, Mycoplasma hyopneumoniae DNA, and porcine circovirus type 2 (PCV2) DNA. A — Mean values of the genomic copy numbers of PRRS RNA in the serum samples. B — Mean values of the genomic copy numbers of M. hyopneumoniae DNA in the nasal swabs, and C — Mean values of the genomic copy numbers of PCV2 DNA in the serum samples in the different groups: Zoetis-VacA/Ch (
), Zoetis-VacB/Ch (
), BI-Vac/Ch (
), UnVac/Ch (
), and UnVac/UnCh (⋄). Different letters within a sampling point mean statistically significant differences (P < 0.05).
Quantification of M. hyopneumoniae DNA in nasal swabs
At the time of challenge, no genomic copies of M. hyopneumoniae were detected in any of the nasal samples from any of the 5 groups. Pigs in UnVac/Ch group had a significantly higher (P < 0.05) number of genomic copies of M. hyopneumoniae in their nasal swabs than pigs in Zoetis-VacA/Ch, Zoetis-VacB/Ch, and BI-Vac/Ch groups at 0 to 56 dpc. Pigs in BI-Vac/Ch group had a significantly higher (P < 0.05) number of genomic copies of M. hyopneumoniae in their nasal swabs than pigs in Zoetis-VacA/Ch and Zoetis-VacB/Ch groups at 0 to 28 dpc. No genomic copies of M. hyopneumoniae were detected in any of the nasal samples from UnVac/UnCh group throughout the study (Figure 2B).
Quantification of PCV2 DNA in blood
At the time of challenge, no genomic copies of PCV2 were detected in any of the serum samples from any of the 5 groups. Pigs in UnVac/Ch group had a significantly higher (P < 0.05) number of genomic copies of PCV2 in their sera than pigs in Zoetis-VacA/Ch, Zoetis-VacB/Ch, and BI-Vac/Ch groups from 7 to 119 dpc. Pigs in BI-Vac/Ch group had a significantly higher (P < 0.05) number of genomic copies of PCV2 in their sera than pigs in Zoetis-VacA/Ch and Zoetis-VacB/Ch groups from 7 to 56 dpc. No genomic copies of PCV2 were detected in any of the serum samples from UnVac/UnCh group throughout the study (Figure 2C).
Immunological responses
Pigs in the Zoetis-VacA/Ch, Zoetis-VacB/Ch, and BI-Vac/Ch groups had significantly higher (P < 0.05) anti-PRRSV antibody levels (Figure 3A) and numbers of PRRSV-specific IFN-γ-SC (Figure 4A) at various dpc compared to pigs in the UnVac/Ch group. No anti-PRRSV antibodies or PRRSV-specific IFN-γ-SC were detected in the UnVac/UnCh group.
Figure 3.
Enzyme-linked immunosorbent assay. A — Mean values of the anti-porcine reproductive and respiratory syndrome virus (PRRSV) antibody levels. B — Mean values of the anti-Mycoplasma hyopneumoniae antibody levels. C — Mean values of the anti-porcine circovirus type 2 (PCV2) antibody levels in the different groups: Zoetis-VacA/Ch (
), Zoetis-VacB/Ch (
), BI-Vac/Ch (
), UnVac/Ch (
), and UnVac/UnCh (⋄). Different letters within a sampling point mean statistically significant differences (P < 0.05).
Figure 4.
Frequency of interferon-γ secreting cells (IFN-γ-SC). A — Mean number of porcine reproductive and respiratory syndrome virus (PRRSV)-specific IFN-γ-SC. B — Mean number of Mycoplasma hyopneumoniae-specific IFN-γ-SC. C — Mean number of PCV2-specific IFN-γ-SC in the different groups: Zoetis-VacA/Ch (
), Zoetis-VacB/Ch (
), BI-Vac/Ch (
), UnVac/Ch (
), and UnVac/UnCh (⋄). Different letters within a sampling point mean statistically significant differences (P < 0.05).
Pigs in the Zoetis-VacA/Ch and Zoetis-VacB/Ch groups had significantly higher (P < 0.05) anti-M. hyopneumoniae antibody levels (Figure 3B) and numbers of M. hyopneumoniae-specific IFNγ-SC (Figure 4B) at various dpc compared to pigs in the BI-Vac/Ch and UnVac/Ch groups. No anti-M. hyopneumoniae antibodies or M. hyopneumoniae-specific IFN-γ-SC were detected in the UnVac/UnCh group.
Pigs in the Zoetis-VacA/Ch and Zoetis-VacB/Ch groups had significantly higher (P < 0.05) anti-PCV2 antibody levels (Figure 3C) at −7 dpc and numbers of PCV2-specific IFN-γ-SC (Figure 4C) at −14, −7, and 0 dpc compared to pigs in the BI-Vac/Ch and UnVac/Ch groups. No anti-PCV2 antibodies or PCV2-specific IFN-γ-SC were detected in the UnVac/UnCh group.
Pathology
Pigs from Zoetis-VacA/Ch, Zoetis-VacB/Ch, and BI-Vac/Ch groups had significantly lower (P < 0.05) scores for macroscopic and microscopic (mycoplasmal pneumonia and interstitial pneumonia) lung lesions, and microscopic lymphoid lesion than pigs from UnVac/Ch group at 119 dpc. Pigs from Zoetis-VacB/Ch group had significantly lower (P < 0.05) scores for macroscopic lung lesions than pigs from BI-Vac/Ch group at 119 dpc. There were no macroscopic lung lesions, and microscopic lung and lymphoid lesions in pigs from UnVac/UnCh group (Table III).
Table III.
Score (mean ± standard deviation) of macroscopic and microscopic lung lesions, porcine reproductive and respiratory syndrome virus (PRRSV) antigen by immunohistochemistry (IHC), Mycoplasma hyopneumoniae (Mhp) DNA by in situ hybridization (ISH), porcine circovirus type 2 (PCV2) antigen by IHC in lung and lymph node in pigs from different groups at 119 d post-challenge (175 d of age).
| Groups | |||||
|---|---|---|---|---|---|
|
|
|||||
| Pathology | Zoetis-VacA/Ch | Zoetis-VacB/Ch | BI-Vac/Ch | UnVac/Ch | UnVac/UnCh |
| Macroscopic lung lesions | 2.72 ± 0.89a,b | 2.39 ± 1.04a | 3.22 ± 0.73b | 10.28 ± 2.22c | 0.83 ± 0.58d |
| Microscopic lung lesions | |||||
| Mycoplasmal pneumonia | 0.67 ± 0.59a | 0.61 ± 0.50a | 0.89 ± 0.68a | 2.50 ± 0.71b | 0.17 ± 0.39c |
| Interstitial pneumonia | 0.72 ± 0.57a | 0.61 ± 0.70a,c | 0.83 ± 0.71a | 2.56 ± 0.78b | 0.08 ± 0.29c |
| Microscopic lymphoid lesions | 0.78 ± 0.73a | 0.67 ± 0.69a,c | 0.89 ± 0.58a | 1.89 ± 0.47b | 0.17 ± 0.39c |
| Lung PRRSV antigen | 4.17 ± 1.42a | 3.94 ± 1.66a | 4.44 ± 1.58a | 6.06 ± 1.59b | 0 ± 0c |
| Lung Mhp nucleic acid | 0.44 ± 0.51a | 0.39 ± 0.50a,c | 0.56 ± 0.51a | 1.11 ± 0.68b | 0 ± 0c |
| Lung PCV2 antigen | 1.78 ± 0.81a | 1.39 ± 0.78a | 1.44 ± 0.70a | 2.67 ± 0.77b | 0 ± 0c |
| Lymphoid PCV2 antigen | 2.06 ± 0.87a | 1.94 ± 0.87a | 2.33 ± 0.69a | 3.39 ± 0.70b | 0 ± 0c |
Different letters mean statistically significant differences (P < 0.05).
Pigs from Zoetis-VacA/Ch, Zoetis-VacB/Ch, and BI-Vac groups had significantly lower (P < 0.05) amounts of PRRSV antigen, M. hyopneumoniae nucleic acid and PCV2 antigen in their lungs compared to pigs from UnVac/Ch group at 119 dpc. Pigs from Zoetis-VacA/Ch, Zoetis-VacB/Ch, and BI-Vac groups had significantly lower (P < 0.05) amounts of PCV2 antigen in their lymph nodes compared to pigs from UnVac/Ch group at 119 dpc. No PRRSV antigen, M. hyopneumoniae nucleic acid, and PCV2 antigen in lung and lymph node was detected in pigs from UnVac/UnCh group at 119 dpc (Table III).
Discussion
The current study shows that the vaccination regimen for PRRSV vaccine, M. hyopneumoniae vaccine, and PCV2 vaccine is efficacious for controlling triple challenge with PRRSV, M. hyopneumoniae, and PCV2 from wean to finish based on clinical, microbiologic, immunologic, and pathologic evaluation. Because PRRSV, M. hyopneumoniae, and PCV2 are now considered important contributors to PRDC (1), this experiment also evaluated PRDC since the animals were challenged with all 3 pathogens. The timing and sequence of infection of these 3 pathogens during challenge mimics natural infection. In Korean pig farms, pigs show severe PRDC signs at 11 to 16 wk after infection with PRRSV and M. hyopneumoniae at 5 to 7 wk followed by PCV2 infection at 7 to 9 wk based on analysis of diagnostic cases and serological survey (C. Chae, personal observation). Hence, vaccinated pigs were challenged with PRRSV and M. hyopneumoniae at 6 wk of age followed by PCV2 at 8 wk of age based on infection patterns in the field. The most striking and consistent microscopic lesions were severe interstitial pneumonia with some degree of peribronchial and peribronchiolar fibrosis consistent with previous reports (1,3,4,22). However, the microscopic lesion scores in the unvaccinated challenged pigs at 119 dpc appear to be lower than those in previous studies at 28 dpc (7,8,27). One reason for this difference could be that a certain degree of the lung lesions in the present study may have been resolved by the time of observation at 119 dpc.
Economic losses by PRDC are mainly due to retardation of growth. Therefore, it is critical to compare growth performance among vaccinated groups. Regardless of commercial vaccines, vaccinated pigs grow significantly more than the unvaccinated pigs. However, a numerical, but not statistically significant reduction of growth performance was observed among 3 vaccinated groups. In addition, retardation of growth is correlated with lung lesions. In the present study, the lung lesions are less severe in the vaccinated pigs than in unvaccinated pigs. Similarly, a reduction in lung lesions is correlated with improved weight gain in previous vaccination studies about PRRSV, M. hyopneumoniae, and PCV2 (28–32). These results indicate that vaccines used in this study help maintain weight gain by reducing lung lesions.
Each vaccine was administered to pigs into separate anatomical sites rather than administering each vaccine in one site, to avoid any interference between vaccines. Ingelvac CircoFLEX and MycoFLEX vaccines have been already been approved for administration as a mixture prior to injection (33). Therefore, administering the mixture of Ingelvac CircoFLEX and MycoFLEX allowed for the comparison of the effects of different vaccination strategies (i.e., mixture of 2 vaccines versus bivalent vaccine) with the Fostera PCV MH (bivalent vaccine of PCV2 and M. hyopneumoniae) in Zoetis-VacB/Ch group under same experimental conditions. Immunological and microbiological results were consistent with the results of 3 vaccination trials in the present study and 2 vaccination trials in previous studies (34–36). Vaccination with mycoplasmal and PCV2 vaccine in Zoetis-VacA/Ch and Zoetis-VacB/Ch groups induces a stronger cellular immune response as measured by frequency of M. hyopneumoniae-and PCV2-specific IFN-γ-SC compared to pigs vaccinated with other vaccines in the BI-Vac/Ch group. Cell-mediated immunity is known to play an important role in preventing mycoplasmal and PCV2 infection (20,37,38). A better cellular immune response seems to play a role in further reducing the amount of M. hyopneumoniae in nasal shedding and the amount of PCV2 in the blood (34–36).
Modified live PRRSV vaccines provide limited protection against heterologous challenge (39). One of the PRRSV challenge strain (lineage 1) used in this study was not closely related to the vaccine viruses, Ingelvac MLV vaccine virus (lineage 5) and Fostera PRRS vaccine virus (lineage 8). However, both modified live PRRSV vaccines were able to reduce the levels of PRRSV viremia. These data suggest that even though the vaccine viruses and the challenge strain are not closely related genetically they may be closely related anti-genically. In addition, genetic similarity does not always guarantee protection, as suggested by a previous study (40).
To our knowledge, this is the first comparative experimental study for the triple vaccination of pigs with PRRSV, M. hyopneumoniae, and PCV2. In particular, for the first time, the bivalent vaccine (PCV2 and M. hyopneumoniae) and monovalent (PRRSV) vaccine in Zoetis-VacB/Ch group were concurrently administered to pigs at separate sites, and the immunological and microbiological results obtained were the same as those obtained by administering each of the 2 monovalent vaccines used in previous studies using same challenging pathogens (17,41). Although medication has been commonly used to control PRDC in most Asian countries, vaccination has increasingly been used as a new cost-effective strategy to control PRDC. The results in this study may provide new options in controlling PRDC, through concurrent administration of single-dose of PRRSV, M. hyopneumoniae, and PCV2 vaccines.
Acknowledgments
This research was supported by contract research funds of the Research Institute for Veterinary Science (RIVS) from the College of Veterinary Medicine and by Brain Korea Plus Program for Creative Veterinary Science Research in the Republic of Korea.
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