Abstract
The objective of this field trial was to compare the effect of 3 different types of combination vaccines on growth performance in pigs under field conditions. The vaccines compared were: a trivalent vaccine containing porcine circovirus type 2a and 2b (PCV-2a/b); and Mycoplasma hyopneumoniae; a mixable bivalent vaccine containing PCV-2a and M. hyopneumoniae; and a ready-to-use bivalent vaccine containing PCV-2a and M. hyopneumoniae. Two farms were selected on the basis of their history of subclinical PCV-2d infection and enzootic pneumonia. A total of 120 pigs on each farm was randomly divided into 4 groups of 30 pigs each. The trivalent-vaccinated group from both farms outperformed each bivalent-vaccinated group in terms of growth performance. Growth performance was significantly improved during the fattening period (70 to 175 d of age) in the mixable bivalent-vaccinated group compared with the ready-to-use bivalent-vaccinated group on 1 farm. The trivalent-vaccinated group elicited higher levels of neutralizing antibodies and interferon-γ secreting cells (IFN-γ-SC) against PCV-2d, while simultaneously decreasing the levels of PCV-2d load in blood when compared with the mixable and ready-to-use bivalent-vaccinated groups. The trivalent-vaccinated group also elicited higher levels of IFN-γ-SC against M. hyopneumoniae and lower levels of M. hyopneumoniae load in the larynx when compared with the mixable and ready-to-use bivalent-vaccinated groups. The results of the present study demonstrated that a trivalent vaccine containing PCV-2a/b and M. hyopneumoniae resulted in a more productive parameter, higher immune responses, and less blood-viral and mycoplasmal larynx-loads when compared with the mixable and ready-to-use bivalent vaccines despite the presence of ongoing subclinical PCV-2d infection and enzootic pneumonia on the farms.
Résumé
L’objectif de cet essai de terrain était de comparer l’effet de trois différents types de vaccins combinés sur les performances de croissance chez les porcs dans des conditions de terrain. Les vaccins comparés étaient : un vaccin trivalent contenant des circovirus porcins de type 2a et 2b (PCV-2a/b) et Mycoplasma hyopneumoniae; un vaccin bivalent mélangeable contenant PCV-2a et M. hyopneumoniae; et un vaccin bivalent prêt à l’emploi contenant le PCV-2a et M. hyopneumoniae. Deux fermes ont été sélectionnées sur la base de leurs antécédents d’infection subclinique par le PCV-2d et de pneumonie enzootique. Un total de 120 porcs dans chaque ferme a été divisé au hasard en quatre groupes de 30 porcs chacun. Le groupe vacciné par les trivalents des deux fermes a surpassé chaque groupe vacciné par les bivalents en termes de performances de croissance. Les performances de croissance ont été significativement améliorées pendant la période d’engraissement (70 à 175 jours d’âge) dans le groupe vacciné bivalent mélangeable par rapport au groupe vacciné bivalent prêt à l’emploi sur une ferme. Le groupe vacciné par trivalent a suscité des niveaux plus élevés d’anticorps neutralisants et de cellules sécrétant de l’interféron-γ (IFN-γ-SC) contre le PCV-2d, tout en diminuant simultanément les niveaux de charge de PCV-2d dans le sang par rapport aux vaccins mélangeables et prêts à l’emploi. Le groupe vacciné trivalent a également provoqué des niveaux plus élevés d’IFN-γ-SC contre M. hyopneumoniae et des niveaux inférieurs de charge de M. hyopneumoniae dans le larynx par rapport aux groupes vaccinés bivalents mélangeables et prêts à l’emploi. Les résultats de la présente étude ont démontré qu’un vaccin trivalent contenant du PCV-2a/b et M. hyopneumoniae entraînait un paramètre plus productif, des réponses immunitaires plus élevées et moins de charges sanguines virales et mycoplasmiques dans le larynx par rapport aux vaccins mélangeables et prêts à l’emploi malgré la présence d’une infection subclinique par le PCV-2d et d’une pneumonie enzootique dans les élevages.
(Traduit par Docteur Serge Messier)
Introduction
Porcine circovirus 2 (PCV-2) and Mycoplasma hyopneumoniae are 2 dominant pathogens that cause costly diseases in the global pork industry. PCV-2 infection can produce overt clinical diseases and porcine circovirus-associated diseases (PCVAD) and cause subclinical PCV-2 infection, the latter considered to be the most common form of PCV-2 infection worldwide (1). PCV-2 is further divided into at least 8 genotypes, noted as “a to h” (2). Among those, PCV-2d is the most predominant genotype in Asia and North America (3–5). Mycoplasma hyopneumoniae infection causes enzootic pneumonia, a chronic respiratory disease that impacts growth (6). Mycoplasma hyopneumoniae continues to frustrate swine practitioners and producers.
Complications from both subclinical PCV-2 infection and enzootic pneumonia in the field are linked to porcine reproductive disease complex (PRDC) that results in economic losses, poor growth performance, and increased costs of treating animals. Vaccination is one of the most efficient tools and is used worldwide to control PCV-2 and M. hyopneumoniae infections. The demand for combination vaccines containing PCV-2 and M. hyopneumoniae has recently increased within the Asian pork industry, as they reduce animal stress and save in labor cost.
Three different types of PCV-2 and M. hyopneumoniae combination vaccines are commercially available: a trivalent vaccine containing PCV-2a/b and M. hyopneumoniae; a mixable bivalent vaccine containing PCV-2a and M. hyopneumoniae; and a ready-to-use bivalent vaccine containing PCV-2a and M. hyopneumoniae. The trivalent vaccine is the first combination vaccine to contain PCV-2b antigen. PCV-2b antigen is so genetically close to PCV-2d that PCV-2d was previously known as mutant PCV-2b. Since combination vaccines containing PCV-2d and M. hyopneumoniae are not yet commercially available, the effect of the 3 different types of existing combination vaccines on growth performance must be compared under field conditions. A full-scale comparative field trial has yet to be undertaken.
The objective of this study was to compare the resulting pig growth performance among 3 different types of combination vaccines in herds with existing subclinical PCV-2 infection and enzootic pneumonia.
Materials and methods
Farm history
The clinical field trial was carried out on 2 farms, noted as Farm A (450 sows) and Farm B (400 sows), located in Chungcheung Province in the Republic of Korea. Both farms were farrow-to-finish operations with an all-in/all-out production system. The porcine reproductive and respiratory syndrome virus (PRRSV) status was stable in the sow population. High-parity (> 5 parity) sows were the only ones seropositive against PRRSV on both farms. Not all sows on both farms were vaccinated against PCV-2 and M. hyopneumoniae.
The 2 farms were selected based on their subclinical PCV-2 infection and enzootic pneumonia status. Subclinical PCV-2 infection was diagnosed on both farms (1) and was defined as follows: decreased average daily weight gain without overt clinical signs, absence of or minimal histopathological lesions in superficial inguinal lymph nodes, and the presence of low amounts of PCV-2 in superficial inguinal lymph nodes (as determined by immunohistochemistry) in 3 out of 4 suspected pigs on Farm A and in 2 out of 5 suspected pigs on Farm B.
Pre-trial investigations identified a PCV-2 serological profile presenting an increase in antibody titers starting around 7 wk of age, whereas 7- to 16-week-old pigs were also PCV-2 polymerase chain reaction (PCR) seropositive on the 2 farms. Mycoplasma hyopneumoniae serology was positive in 12- to 16-week-old pigs. Furthermore, the nasal swabs of 7-week-old pigs were PCR-positive for M. hyopneumoniae on the 2 farms. Together these results supported an active PCV-2 and M. hyopneumoniae infection on Farms A and B.
Experimental design
To minimize sow variation, at 21 d of age, 8 piglets were pulled from each of 15 sows and uniformly divided into 4 groups (2 per group). A total of 120 pigs on each farm was used in this comparative field trial and were randomly assigned into 1 of 4 groups (30 pigs per group, male = 15 and female = 15) using the random number generator function (Excel; Microsoft Corporation, Redmond, Washington, USA) (Table I).
Table I.
Field experimental design.
| Group | Type of vaccine | Name of vaccine |
|---|---|---|
| VacA1 and VacB1 | Ready-to-use trivalent | Fostera Gold PCV MH |
| VacA2 and VacB2 | Mixable bivalent | Ingelvac CircoFLEX + MycoFLEX |
| VacA3 and VacB3 | Ready-to-use bivalent | Porcilis PCV M Hyo |
| UnVacA and UnVacB | None | None |
Pigs in the VacA1 and VacB1 groups received a 2.0 mL dose of trivalent vaccine (Fostera Gold PCV MH, Serial No. 395164A, Expiration date: 10-Dec-2021; Zoetis, Parsippany, New Jersey, USA) by intramuscular route (IM) in the neck muscle at 21 d of age. Pigs in the VacA2 and VacB2 groups received a 2.0 mL dose of freshly mixed bivalent vaccine (FLEXcombo; Boehringer Ingelheim Vetmedica, St. Joseph, Missouri, USA) IM in the neck muscle at 21 d of age. FLEXcombo vaccine was prepared by mixing equal volumes of Ingelvac CircoFLEX (Serial No. 3091337B, Expiration date: 15-Apr-2021) and Ingelvac MycoFLEX (Serial No. 2730677A, Expiration date: 10-Dec-2021) prior to use according to label directions. Pigs in the VacA3 and VacB3 groups received a 2.0 mL dose of bivalent vaccine (Porcilis PCV M Hyo, Lot No. C752B02, Expiration date: 23-Sep-2021; MSD Animal Health, Boxmeer, The Netherlands) IM in the neck muscle at 21 d of age. Pigs in the UnVacA and UnVacB groups received 2.0 mL of phosphate-buffered saline (PBS, 0.01M, pH 7.4) IM at 21 d of age.
Blood and laryngeal swabs were collected from each treatment group at 0 (21 d old), 28 (49 d old), 49 (70 d old), and 91 (112 d old) days post-vaccination (dpv). Pigs were snared and restrained with a mouth gag for laryngeal swab collection, during which the swabs were guided into the larynx with a laryngoscope. Once the epiglottis was in a low position, the internal walls of the laryngeal cartilages were swept with the swabs.
Clinical observations
The pigs were monitored daily for abnormal clinical signs and scored (0 to 6) weekly for severity of clinical respiratory signs with a blind system (7). Mortality and age of death were recorded. Pigs that died during the field trial were necropsied to diagnose the cause of death.
Average daily weight gain
The pigs were weighed on day 0 (21 d old), 49 (70 d old), and 154 (175 d old) dpv. The average daily weight gain (ADWG; g/pig/d) was analyzed over 2 time periods: i) between 21 and 70 d old; and ii) between 70 and 175 d old. The ADWG was calculated as the difference between the body weights of 2 weight time points divided by the number of days between these 2 weight time points. Data for dead or removed pigs were included in the calculation.
Quantification of PCV-2d DNA in blood
DNA was extracted from serum samples using a commercial kit (QIAamp DNA Mini Kit; QIAGEN, Valencia, California, USA). Real-time polymerase chain reaction (RT-PCR) was carried out to quantify the amount of PCV-2d genomic DNA (8).
Quantification of M. hyopneumoniae DNA in laryngeal swabs
DNA was extracted from laryngeal swabs using a commercial kit (QIAamp DNA Mini Kit; QIAGEN). Real-time PCR was carried out to quantify the amount of the M. hyopneumoniae genomic DNA (9).
Serology
The serum samples were tested using commercially available enzyme-linked immunosorbent assays (ELISAs) for PCV-2 (SERELISA PCV-2 Ab Mono Blocking; Synbiotics, Lyon, France) and M. hyopneumoniae (M. hyo. Ab test; IDEXX Laboratories, Westbrook, Maine, USA). For ELISA results, serum samples were considered positive for antibodies against PCV-2 if the reciprocal ELISA titer was > 350 and positive for antibodies against M. hyopneumoniae if the sample-to-positive (S/P) ratio was ≥ 0.4, in accordance with the manufacturer’s instructions for each kit. The serum samples were also tested to measure neutralizing antibody (NA) titers against PCV-2d (10–12).
Enzyme-linked immunospot assay
Enzyme-linked immunospot (ELISpot) assay was conducted to determine the frequency of PCV-2d- and M. hyopneumoniae-specific interferon-γ secreting cells (IFN-γ-SCs) in peripheral blood mononuclear cells (PBMCs) (8,13). The IFN-γ positive spots on the membranes were imaged, analyzed, and counted using an automated ELISpot Reader (AID ELISPOT Reader; AID GmbH, Strassberg, Germany). The frequency of PCV-2d- and M. hyopneumoniae-specific IFN-γ-SCs was expressed as the number of IFN-γ-SCs per million PBMC. ELISpot assay was done in duplicate.
Pathology
Macroscopic lung lesion scores were evaluated by the percentage of the pneumonic lesions. The scoring was carried out and recorded by 2 pathologists (Chae and 1 graduate student). 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 (7). Microscopic lung mycoplasmal lesions were scored (0 to 6) based on the severity of peribronchiolar lymphoid tissue hyperplasia (14). Microscopic lymphoid lesions were scored (0 to 5) based on the severity of lymphoid depletion and granulomatous inflammation (15).
Statistical analysis
Prior to statistical analysis, real-time PCR data were transformed to log10 values. The Shapiro-Wilk test was used to check the collected data for a normal distribution. If the normality assumption was met, 1-way analysis of variance (ANOVA) was conducted. Results from the ANOVA test that showed statistical significance were further evaluated by 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. Kruskal-Wallis test results with a statistical significance were further evaluated with the Mann-Whitney test to include Tukey’s adjustment to compare the differences among the groups. Results were reported in P-value, with P < 0.05 considered significant.
Results
Clinical signs
On Farm A, the vaccinated group (VacA1) had significantly lower (P < 0.05) respiratory signs than those of the unvaccinated group (UnVacA) at 21 and 84 to 112 dpv. All 3 vaccinated groups (VacA1, VacA2, and VacA3) had significantly lower (P < 0.05) respiratory signs than those of the unvaccinated group (UnVacA) at 28 to 77 dpv.
On Farm B, 1 vaccinated group (VacB1) had significantly lower (P < 0.05) respiratory signs than those of the unvaccinated group (UnVacB) at 14 and 91 to 133 dpv. The vaccinated groups (VacB1, VacB2, and VacB3) had significantly lower (P < 0.05) respiratory signs than those of the unvaccinated group (UnVacB) at 21 to 84 dpv.
Average daily weight gain
No difference in mean body weight was observed between vaccinated and unvaccinated animals at the time the study began (21 d of age) on either farm. On Farm A, the average daily weight gain (ADWG) of vaccinated animals (VacA1, VacA2, and VacA3 groups) was significantly higher (P < 0.05) than that of unvaccinated animals (UnVacA group) during the fattening (70 to 175 d of age) and overall (21 to 175 d) periods. The ADWG of the VacA1 group was significantly higher (P < 0.05) than that of the VacA2 and VacA3 groups during the fattening period (70 to 175 d of age) (Figure 1 A).
Figure 1.
Average daily weight gain (ADWG) on Farm A (A) from VacA1 (
), VacA2 (
), VacA3 (
), and UnVacA (
) and on Farm B (B) from VacB1 (
), VacB2 (
), VacB3 (
), and UnVacB (
). Variation is expressed as the standard deviation.
a,b,c Indicate significant (P < 0.05) differences among 4 groups on each farm.
The ADWG of vaccinated animals (VacB1, VacB2, and VacB3 groups) on Farm B was significantly higher (P < 0.05) than that of unvaccinated animals (UnVacB group) during the fattening period (70 to 175 d of age) and overall period (21 to 175 d). The ADWG of the VacB1 and VacB2 groups was significantly higher (P < 0.05) than that of the VacB3 group during the fattening period (70 to 175 d of age) (Figure 1 B).
Mortality
Mortality on Farm A was reported as follows. Two pigs from the VacA1 group died at 55 and 74 d of age, respectively, of suppurative bronchopneumonia as diagnosed by a combination of M. hyopneumoniae that was detected with PCR and Trueperella pyogenes that was isolated from the lungs. One pig from the VacA2 group died of severe watery diarrhea at 70 d of age, in which bacteria was not found during isolation from the small and large intestine. One pig from the VacA2 group died at 85 d of age of bronchopneumonia as diagnosed by a combination of M. hyopneumoniae that was detected with PCR and Pasteurella multocida that was isolated from the lungs. One pig from the VacA3 died at 73 d of age of bronchopneumonia and pleuritis as diagnosed by a combination of PCV-2d and M. hyopneumoniae that was detected with PCR and Glaesserella parasuis that was isolated from the lungs.
Three pigs from the UnVacA group died at 65, 72, and 88 d of age, respectively, of bronchopneumonia as diagnosed by a combination of PCV-2d and M. hyopneumoniae that was detected with PCR and P. multocida that was isolated from the lungs. One pig died at 78 d of age of bronchopneumonia as diagnosed by a combination of M. hyopneumoniae that was detected with PCR and P. multocida that was isolated from the lungs.
Mortality on Farm B was reported as follows. One pig from the VacB1 group died at 65 d of age of unknown etiology without any pathological lesions in lung and brain. One pig from the VacB2 group died at 77 d of age of bronchopneumonia as diagnosed by a combination of PCV-2d that was detected with PCR and G. parasuis that was isolated from the lungs. Two pigs from the VacB3 group died at 72 d of age of bronchopneumonia as diagnosed by a combination of M. hyopneumoniae that was detected with PCR and T. pyogenes that was isolated from the lungs. One pig from the VacB3 group died at 80 d of age of bronchopneumonia and fibrinous pericarditis as diagnosed by a combination of PCV-2d that was detected with PCR and G. parasuis that was isolated from the lungs and pericardium.
One pig from the UnVacB group died at 74 d of age of unknown etiology without any pathological lesions in lung and brain. Two pigs from the UnVacB group died at 77 and 83 d of age as diagnosed by a combination of PCV-2d and M. hyopneumoniae that were detected with PCR and P. multocida that was isolated from the lungs.
Quantification of PCV-2d in blood
On Farm A, vaccinated animals (VacA1, VacA2, and VacA3 groups) had significantly lower (P < 0.05) amounts of PCV-2d load in their blood than those of unvaccinated animals (UnVacA group) at 28, 49, and 91 dpv (Figure 2 A). On Farm B, vaccinated animals (VacB1, VacB2, and VacB3 groups) had significantly lower (P < 0.05) amounts of PCV-2d load in their blood than those of unvaccinated animals (UnVacB group) at 28, 49, and 91 dpv. The amount of PCV-2d load in blood from the VacB1 group was significantly lower (P < 0.05) than that of the VacB3 group at 49 dpv (Figure 2 B).
Figure 2.
Genomic copy number of porcine circovirus 2d (PCV-2d) and Mycoplasma hyopneumoniae DNA. A — Mean values of the genomic copy number of PCV-2d in blood from VacA1 (
), VacA2 (
), VacA3 (
), and UnVacA (
) groups on Farm A. B — Mean values of the genomic copy number of PCV-2d in blood from VacB1 (
), VacB2 (
), VacB3 (
), and UnVacB (
) groups on Farm B. C — Mean values of the genomic copy number of M. hyopneumoniae in larynx from VacA1 (
), VacA2 (
), VacA3 (
), and UnVacA (
) groups in Farm A. D — Mean values of the genomic copy number of M. hyopneumoniae in larynx from VacB1 (
), VacB2 (
), VacB3 (
), and UnVacB (
) groups on Farm B. Variation is expressed as the standard deviation.
a,b,c Indicate significant (P < 0.05) differences among 4 groups on each farm.
Quantification of M. hyopneumoniae in laryngeal swabs
On Farm A, vaccinated animals (VacA1, VacA2, and VacA3 groups) had significantly lower (P < 0.05) amounts of M. hyopneumoniae load in their larynx than those of unvaccinated animals (UnVacA group) at 28, 49, and 91 dpv (Figure 2 C).
On Farm B, vaccinated animals (VacB1, VacB2, and VacB3 groups) had significantly lower (P < 0.05) amounts of M. hyopneumoniae load in their larynx than those of unvaccinated animals (UnVacB group) at 28, 49, and 91 dpv. The amount of M. hyopneumoniae load in the larynx from the VacB1 group was significantly lower (P < 0.05) than in the VacB2 and VacB3 groups at 49 dpv (Figure 2 D).
Immune responses against PCV-2
On Farm A, vaccinated animals (VacA1, VacA2, and VacA3 groups) had significantly higher (P < 0.05) PCV-2 ELISA and NA titers than those of unvaccinated animals (UnVacA group) at 28, 49, and 91 dpv. A comparison of vaccinated groups showed that PCV-2 ELISA (Figure 3 A) and NA titers (Figure 3 C) from the VacA1 group were significantly higher (P < 0.05) than those of the VacA3 group at 91 dpv. Vaccinated animals (VacA1, VacA2, and VacA3 groups) had significantly higher (P < 0.05) PCV-2d-specific IFN-γ-SC levels than unvaccinated animals (UnVacA group) at 28, 49, and 91 dpv. A comparison of vaccinated groups showed that the VacA1 group PCV-2d-specific IFN-γ-SC level was significantly higher (P < 0.05) than that of VacA3 groups at 49 and 91 dpv (Figure 3 E).
Figure 3.
Immune responses against porcine circovirus 2d (PCV-2d). A — Mean values of PCV-2 enzyme-linked immunosorbent assay (ELISA) in serum from VacA1 (
), VacA2 (
), VacA3 (
), and UnVacA (
) groups on Farm A. B — Mean values of the PCV-2 ELISA in serum from VacB1 (
), VacB2 (
), VacB3 (
), and UnVacB (
) groups on Farm B. C — Mean values of the neutralizing antibody (NA) titers against PCV-2d in serum from VacA1 (
), VacA2 (
), VacA3 (
), and UnVacA (
) groups on Farm A. D — Mean values of the NA titers against PCV-2d in serum from VacB1 (
), VacB2 (
), VacB3 (
), and UnVacB (
) groups on Farm B. E — Mean values of the PCV-2d-specific interferon-γ secreting cells (IFN-γ-SCs)/106 peripheral blood mononuclear cells (PBMCs) from VacA1 (
), VacA2 (
), VacA3 (
), and UnVacA (
) groups on Farm A. F — Mean values of the PCV-2d-specific IFN-γ-SCs/106 PBMCs from VacB1 (
), VacB2 (
), VacB3 (
), and UnVacB (
) groups on Farm B. Variation is expressed as the standard deviation.
a,b,c Indicate significant (P < 0.05) differences among 4 groups on each farm.
On Farm B, vaccinated animals (VacB1, VacB2, and VacB3 groups) had significantly higher (P < 0.05) PCV-2 ELISA (Figure 3 B) and NA titers (Figure 3 D) than unvaccinated (UnVacB group) animals at 28, 49, and 91 dpv. In a comparison of vaccinated groups, PCV-2 ELISA titers from the VacB1 group were significantly higher (P < 0.05) than those of the VacB3 group at 91 dpv (Figure 3 B). PCV-2 NA titers from the VacB1 group were significantly higher (P < 0.05) than those of the VacB3 group at 49 and 91 dpv (Figure 3 D).
Vaccinated animals (VacB1, VacB2, and VacB3 groups) had significantly higher (P < 0.05) PCV-2d-specific IFN-γ-SC levels than unvaccinated (UnVacB group) animals at 28, 49, and 91 dpv. In a comparison of vaccinated groups, the PCV-2d-specific IFN-γ-SC level from the VacB1 group was significantly higher (P < 0.05) than that of VacB3 groups at 28, 49, and 91 dpv (Figure 3 F).
Immune responses against M. hyopneumoniae
On Farm A, vaccinated animals (VacA1, VacA2, and VacA3 groups) had significantly higher (P < 0.05) M. hyopneumoniae ELISA S/P ratios than unvaccinated animals (UnVacA group) at 28, 49, and 91 dpv. Among the vaccinated groups, M. hyopneumoniae ELISA S/P ratios from the VacA1 group were significantly higher (P < 0.05) than those of the VacA3 group at 49 and 91 dpv (Figure 4 A).
Figure 4.
Immune responses against Mycoplasma hyopneumoniae. A — Mean values of the M. hyopneumponiae enzyme-linked immunosorbent assay (ELISA) in serum from VacA1 (
), VacA2 (
), VacA3 (
), and UnVacA (
) groups on Farm A. B — Mean values of the M. hyopneumponiae ELISA in serum from VacB1 (
), VacB2 (
), VacB3 (
), and UnVacB (
) groups on Farm B. C — Mean values of the M. hyopneumoniae-specific interferon-γ secreting cells (IFN-γ-SCs)/106 peripheral blood mononuclear cells (PBMCs) from VacA1 (
), VacA2 (
), VacA3 (
), and UnVacA (
) groups on Farm A. D — Mean values of the M. hyopneumoniae-specific IFN-γ-SCs/106 PBMCs from VacB1 (
), VacB2 (
), VacB3 (
), and UnVacB (
) groups on Farm B. Variation is expressed as the standard deviation.
a,b,c Indicate significant (P < 0.05) differences among 4 groups on each farm.
Vaccinated animals (VacA1, VacA2, and VacA3 groups) had significantly higher (P < 0.05) M. hyopneumoniae-specific IFN-γ-SC levels than unvaccinated animals (UnVacA group) at 28, 49, and 91 dpv. In a comparison of vaccinated groups, M. hyopneumoniae-specific IFN-γ-SC levels from the VacA1 group were significantly higher (P < 0.05) than those of the VacA3 group at 28 and 49 dpv (Figure 4 C).
On Farm B, vaccinated animals (VacB1, VacB2, and VacB3 groups) had significantly higher (P < 0.05) M. hyopneumoniae ELISA S/P ratios than unvaccinated animals (UnVacB group) at 28, 49, and 91 dpv. In a comparison of vaccinated groups, M. hyopneumoniae ELISA S/P ratios from the VacB1 group were significantly higher (P < 0.05) than those of the VacB3 group at 91 dpv (Figure 4 B).
Vaccinated animals (VacB1, VacB2, and VacB3 groups) had significantly higher (P < 0.05) M. hyopneumoniae-specific IFN-γ-SC levels than unvaccinated animals (UnVacB group) at 28, 49, and 91 dpv. Among vaccinated groups, the M. hyopneumoniae-specific IFN-γ-SC level from the VacB1 group was significantly higher (P < 0.05) than that of the VacB3 group at 49 dpv (Figure 4 D).
Pathology
Vaccinated animals (VacA1, VacA2, VacA3, VacB1, VacB2, and VacB3 groups) at Farms A and B had significantly lower (P < 0.05) macroscopic and microscopic lung lesion scores than those of unvaccinated animals (UnVacA and UnVacB groups) at 154 dpv (Table II).
Table II.
Macroscopic and microscopic pathology of vaccinated and unvaccinated groups at 154 dpv.
| Farm | Group | Macroscopic Lung lesion | Microscopic | |
|---|---|---|---|---|
|
| ||||
| Lung lesion | Lymphoid lesion | |||
| Farm A | VacA1 | 15.54 ± 6.00a | 0.78 ± 0.55a | 0.69 ± 0.49 |
| VacA2 | 16.99 ± 5.54a | 0.75 ± 0.56a | 0.76 ± 0.56 | |
| VacA3 | 17.30 ± 7.73a | 0.86 ± 0.52a | 0.93 ± 0.83 | |
| UnVacA | 27.70 ± 9.79b | 2.12 ± 0.91b | 1.03 ± 0.35 | |
| Farm B | VacB1 | 16.72 ± 5.25a | 0.73 ± 0.60a | 0.74 ± 0.59 |
| VacB2 | 16.48 ± 4.36a | 0.79 ± 0.53a | 0.79 ± 0.58 | |
| VacB3 | 18.39 ± 6.57a | 0.81 ± 0.50a | 0.84 ± 0.51 | |
| UnVacB | 30.94 ± 10.7b | 2.47 ± 1.04b | 1.03 ± 0.38 | |
Indicate significant (P < 0.05) differences among 4 groups at each farm.
Discussion
Two farms that represented typical Korean pig farms in terms of housing conditions and health status were selected for this study on the basis of their history with subclinical PCV-2d infection and enzootic pneumonia. As retardation of growth was a common clinical feature of these 2 diseases at the farms, growth performance was the most critical protective index when comparing the 3 combination vaccines.
In the present comparative field trial, the trivalent-vaccinated pigs had better growth performance than pigs in the mixable and ready-to-use bivalent-vaccinated groups. The mixable bivalent vaccine was able to significantly improve the growth performance during the fattening period (70 to 175 d of age) compared to the ready-to-use bivalent vaccine. These results are in contrast with a comparative experimental challenge study (16) in which statistical differences in growth performance were not observed between the same 2 bivalent vaccines.
Several possibilities may account for the discrepancy between experimental and field conditions. Pig-rearing conditions in experimental studies are different from field conditions, under which pigs continue to expose and re-expose themselves to field PCV-2d and M. hyopneumoniae by horizontal and vertical transmission, which exacerbates the disease. Other key differences include the small sample size (number of animals per group), short duration of the study, and the overly regulated experimental conditions, all of which are different from commercial farms. Several key factors affect pig growth performance in field conditions, such as herd characteristics, biosecurity, and husbandry practices and management. When all of these factors are combined, comparative field clinical trials are able to determine exactly what type of vaccines yield better growth performance.
All 3 types of combination vaccines evaluated in this trial successfully induced the protective immunity of pigs as seen by humoral and cellular immune responses throughout the course of the study. A higher level of protective immunity results in the reduction of pathogen loads in tissues and pathological lesions, which consequently protects pigs from the pathogens. Types of protective immunity against PCV-2, such as neutralizing antibodies and IFN-γ-SC levels, are well-correlated with the reduction of PCV-2 loads in blood and the reduction of lymphoid lesions (11,17–19).
The trivalent-vaccinated group elicited higher levels of PCV-2d neutralizing antibodies and IFN-γ-SCs and lower levels of PCV-2d load in blood than the mixable and ready-to-use bivalent-vaccinated groups. Although the trivalent vaccine outperformed both bivalents in terms of immune response and viral load, statistical differences in lymphoid lesions were not observed among any of the 3 vaccinated or unvaccinated groups. This may be attributed to the fact that both farms had concurrent subclinical PCV-2 infection circulating within the herds. These results may be due to subclinical PCV-2 infection, which results in mild lymphoid lesions or even the absence of lymphoid lesions altogether.
Although protective immunity against M. hyopneumoniae is not well-defined, cell-mediated immunity is used to evaluate pig protection from M. hyopneumoniae infection (20,21). The trivalent vaccine outperformed the mixable and ready-to-use bivalent vaccines for M. hyopneumoniae protection, as indicated by the higher levels of elicited IFN-γ-SCs and lower levels of M. hyopneumoniae load in the larynx than those of the vaccinated group. A statistical difference was not observed among any of the vaccinated groups in relation to lung lesion severity, which indicates that cell-mediated immunity may not be the only protective immunity.
Increased growth performance, higher immune responses, and lower viral and mycoplasmal loads between the trivalent and 2 (mixable and ready-to-use) bivalent vaccines may be attributed to the difference in antigens and adjuvant among vaccines. The trivalent vaccine was the only vaccine containing PCV-2b antigen. Combination vaccines containing PCV-2b antigen may be clinically superior to other combination vaccines that contain PCV-2a antigen in the efficient control of PCV-2d infection due to the genetic proximity between PCV-2b and PCV-2d (22). The M. hyopneumoniae antigen strain in the trivalent vaccine differed from that in the 2 mixable and ready-to-use bivalent vaccines that both shared the same strain (22). It is therefore possible that the different strain of M. hyopneumoniae antigen found in the trivalent vaccine may have affected its immunogenicity and protective effect more than the strain in the bivalent vaccines.
The clinical field data gathered from this study is intended to provide pig producers with clinical information about growth performance. Complications of both subclinical PCV-2 infection and enzootic pneumonia appear to cause porcine respiratory disease complex (PRDC) in the field. The present study demonstrated that a trivalent vaccine containing PCV-2a/b and M. hyopneumoniae resulted in a better productive parameter and immune responses, lower viral blood load, and reduced mycoplasmal larynx load when compared with 2 different bivalent vaccines in the presence of ongoing herd subclinical PCV-2d infection and enzootic pneumonia.
Acknowledgments
The authors’ research was supported by contract research funds (Grant no. 550-20190068) from the Research Institute for Veterinary Science (RIVS) at the College of Veterinary Medicine and the BK 21 FOUR Future Veterinary Medicine Leading Education and Research Center (Grant no. A0449-20200100).
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