Abstract
The objective of this study was to compare the efficacy of 2 different commercial Mycoplasma hyopneumoniae vaccines and porcine reproductive and respiratory syndrome virus (PRRSV) vaccines in regard to growth performance, microbiological and immunological analyses, and pathological observation from wean to finish (175 d of age). Pigs were administered M. hyopneumoniae and PRRSV vaccines at 7 and 21 d of age, respectively, or both at 21 d old and then challenged with both M. hyopneumoniae and PRRSV at 49 d old. Significant (P < 0.05) differences were observed between the 2 vaccinated challenged groups in average daily weight gain, nasal shedding of M. hyopneumoniae, M. hyopneumoniae-specific interferon-γ secreting cells, and macroscopic and microscopic lung lesions. Induction of interleukin-10 following PRRSV vaccination does not interfere with the immune responses induced by M. hyopneumoniae vaccine. The present study demonstrated that the single-dose vaccination regimen for M. hyopneumoniae and PRRSV vaccine is efficacious for controlling coinfection with M. hyopneumoniae and PRRSV based on clinical, microbiological, immunological, and pathological evaluation.
Résumé
L’objectif de la présente étude était de comparer l’efficacité de deux vaccins commerciaux différents contre Mycoplasma hyopneumoniae et le virus du syndrome reproducteur et respiratoire porcin (VSRRP) quant aux performances de croissance, aux analyses microbiologiques et immunologiques, et les observations pathologiques chez des porcs du sevrage à la finition (175 j d’âge). Les porcs ont reçu les vaccins M. hyopneumoniae et VSRRP à 7 et 21 j d’âge, respectivement, ou les deux à 21 j et par la suite soumis à une infection défi avec M. hyopneumoniae et VSRRP à l’âge de 49 j. Des différences significatives (P < 0,05) ont été observées entre les deux groupes vaccinés et challengés pour les paramètres suivants : le gain quotidien moyen, l’excrétion nasale de M. hyopneumoniae, le nombre de cellules secrétant de l’interféron-γ spécifique à M. hyopneumoniae, et les lésions pulmonaires macroscopiques et microscopiques. L’induction d’interleukine-10 suivant la vaccination pour VSRRP n’a pas interférée avec les réponses immunitaires induites par le vaccin M. hyopneumoniae. Cette étude a démontré qu’une vaccination avec une dose unique de vaccin contre M. hyopneumoniae et le VSRRP est efficace pour limiter une co-infection par ces deux agents si on se base sur les évaluations clinique, microbiologique, immunologique et pathologique.
(Traduit par Docteur Serge Messier)
Introduction
Mycoplasma hyopneumoniae is the etiological pathogen of enzootic pneumonia, which is characterized by a chronic, nonproductive cough (1). Infection of M. hyopneumoniae causes considerable economic losses, due to decreased growth rates, high feed conversion ratios, increased medication costs, and the susceptibility of sick pigs to infection by other organisms (1,2). Porcine reproductive and respiratory syndrome (PRRS) virus (PRRSV) is an enveloped, single-stranded, positive-sense RNA virus belonging to the Arteriviridae family in the order Nidovirales (3) that can cause reproductive problems in sows and respiratory problems in growing pigs (4).
In pigs, respiratory disease is multifactorial and complex and is caused by sequential or concurrent infections with several viral or bacterial pathogens; therefore, the name porcine respiratory disease complex (PRDC) is used to describe this disease (5,6). The economic impact of PRDC is tremendous, mainly due to decreased fattening performance and the cost of medication (7,8). Currently, the use of antibiotics for controlling PRDC is limited due to increased risk of antimicrobial resistance and residue in carcasses (9). Therefore, vaccinations are of prime importance and are routinely applied worldwide.
Since coinfection with M. hyopneumoniae and PRRSV is one of the most economically important situations in PRDC (10), vaccination of pigs with both M. hyopneumoniae and PRRSV is necessary to control PRDC efficiently. The commercial modified live PRRSV vaccine (Ingelvac PRRS MLV; Boehringer Ingelheim Vetmedica, St. Joseph, Missouri, USA) was first licensed for worldwide use in 1994. In 2012, another new commercial modified live PRRSV vaccine (Fostera PRRS; Zoetis, Florham Park, New Jersey, USA) was introduced to the international market to control respiratory disease in growing pigs. A comparison of both single-dose M. hyopneumoniae and PRRSV vaccines together, therefore, is more practical and mirrors field conditions, rather than a comparison of each single dose M. hyopneumoniae and PRRSV vaccines by themselves. The objective of the present study was to compare the efficacy of 2 commercial single-dose M. hyopneumoniae vaccines and PRRSV vaccines in regard to virological and immunological analysis, pathological observation, and growth performance from wean to finish using a challenge model.
Materials and methods
Commercial vaccines
Two types of commercial M. hyopneumoniae vaccines were used in this study: A — the inactivated M. hyopneumoniae bacterin (RespiSure-One; Zoetis) given as one 2.0-mL dose at 7 d of age and B — the inactivated M. hyopneumoniae bacterin (Ingelvac MycoFLEX; Boehringer Ingelheim Vetmedica) given as one 1.0-mL dose at 21 d of age. Two types of commercial PRRSV vaccines were used in this study: A — the modified live PRRSV vaccine (Fostera PRRS; Zoetis) given as one 2.0-mL dose at 21 d of age, and B — the modified live PRRSV vaccine (Ingelvac PRRS MLV; Boehringer Ingelheim Vetmedica) given as one 2.0-mL dose at 21 d of age. 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
Mycoplasma hyopneumoniae strain SNU98703, used as inoculum, was isolated from lung samples from postweaned pigs with severe enzootic pneumonia (11). Mycoplasma hyopneumoniae strain SNU98703 caused typical lesions consisting of peribronchial, peribronchiolar, and perivascular lymphoid hyperplasia in experimentally infected pigs (11). The PRRSV strain SNUVR090851 (type 2 genotype, lineage 1, GenBank no. JN315685), used as inoculum, was isolated from lung samples from different newly weaned pigs in Chungcheung Providence in 2010 (12). This virus strain caused interstitial pneumonia characterized by thickened alveolar septa with increased numbers of interstitial macrophages and lymphocytes and by type II pneumocyte hyperplasia in experimentally infected pigs (12).
Animals
A total of 60 colostrum-fed, cross-bred, conventional piglets were purchased at 5 d of age from an M. hyopneumoniae- and PRRSV-free commercial farm, based on serological testing of breeding herd and long-term clinical and slaughter history. All piglets were negative for M. hyopneumoniae, PRRSV, and porcine circovirus type 2 (PCV2) according to routine serological testing. Mycoplasma hyopneumoniae was not detected in the nasal samples by the real-time polymerase chain reaction (PCR) (13). The PRRSV and PCV2 were not detected in the serum and nasal samples by the real-time polymerase chain reaction (PCR) (14,15).
Experimental design
A total of 60 pigs were randomly divided into 4 groups: Zoetis-Vac (n = 20 pigs), BI-Vac (n = 20 pigs), positive control (n = 10), and negative control (n = 10) groups using the random number generation function (Excel; Microsoft Corporation, Redmond, Washington, USA). In the Zoetis-Vac group, pigs were immunized with the M. hyopneumoniae A bacterin and the PRRSV A vaccines at 7 and 21 d of age, respectively. In BI-Vac group, pigs were immunized with both the M. hyopneumoniae B bacterin (left side of the neck) and PRRSV B vaccine (right side of the neck) at 21 d of age. At 49 d of age [0 days post challenge (dpc)], pigs in the Zoetis-Vac, BI-Vac, and positive control groups were intratracheally administered a 10 mL dose of a lung homogenate of M. hyopneumoniae strain SNU98703 (1:100 dilution in Friis medium) at a final concentration of 104 to 105 color-changing units (CCU)/mL in the morning, as previously described (16). In the afternoon of the same day, same pigs were intranasally administered 3 mL of type 2 PRRSV (strain SNUVR090851; 2nd passage in MARC-145 cells) containing 1.2 × 105 TCID50/mL. Blood samples from each pig were collected by jugular venipuncture at −42, −28, 0, 14, 28, 63, 91, and 126 dpc. Sterile polyester swabs (Fisher Scientific, Pittsburgh, Pennsylvania, USA) were used to swab the nasal mucosa of both nostrils, reaching deeply into the turbinates at −42, −28, 0, 14, 28, 63, 91, and 126 dpc. Swabs were stored in 5 mL plastic tubes (Fisher Scientific) containing 1 mL of sterile saline solution. Pigs were sedated with an intravenous injection of azaperon (Stresnil; Janssen Pharmaceutica, Beerse, Belgium) and then euthanized by electrocution for necropsy at 175 d of age (126 dpc). Tissues were collected from each pig at necropsy. All of the methods were approved by the Seoul National University Institutional Animal Care and Use Committee.
Clinical evaluation
Following M. hyopneumoniae and PRRSV inoculation, the pigs were monitored daily for physical condition and scored weekly for clinical respiratory disease severity using scores ranging from 0 (normal) to 6 (severe dyspnea and abdominal breathing) (17). Observers were blinded to vaccination and challenge status. Pigs were observed daily at the same time of day. Rectal body temperature was recorded daily from 0 to 21 dpc.
Assessment of growth performance
The live weight of each pig was measured at 3 (−28 dpc), 7 (0 dpc), 10 (21 dpc), 16 (63 dpc), and 25 (126 dpc) weeks of age. The average daily weight gain (ADWG, grams/pig per day) was analyzed over 4 time periods: between 3 and 7; 7 and 10; 10 and 16; and 16 and 25 weeks of age. The ADWG during these various production stages was calculated as the difference between the starting and final weights divided by the duration of the stage. Data from dead pigs were included in the calculation.
Serology
The serum samples were tested using the commercially available M. hyopneumoniae and PRRSV enzyme-linked immunosorbent assay (ELISA; IDEXX M.hyo Ab Test and PRRS X3 Ab Test, IDEXX Laboratories, Westbrook, Maine, USA). Serum virus neutralization tests for PRRSV were also done using the challenge strain, as previously described (18,19). Serum samples were considered to be positive for neutralizing antibodies (NA) if the titer was greater than 2.0 (log2) (20).
Quantification of M. hyopneumoniae DNA
DNA was extracted from the nasal swabs using a kit (QIAamp DNA Mini Kit; QIAGEN, Crawley, United Kingdom). The DNA extracts were then used to quantify the M. hyopneumoniae genomic DNA copy numbers by real-time PCR, as previously described (13).
Quantification of PRRSV RNA
RNA was extracted from serum samples and nasal swabs using a kit (QIAamp Viral RNA Mini Kit; QIAGEN) to quantify type 2 PRRSV genomic cDNA copy numbers by real-time PCR, as previously described (14).
Enzyme-linked immunospot assay
Mycoplasma hyopneumoniae and PRRSV antigens were prepared as previously described (21,22). The numbers of M. hyopneumoniae- and PRRSV-specific interferon-γ secreting cells (IFN-γ-SC) stimulated by challenging M. hyopneumoniae and PRRSV were determined in peripheral blood mononuclear cells (PBMC) as previously described (23–25). The results were expressed as the numbers of IFN-γ-SC per million PBMC.
Quantification of interleukin-10 secretion
The interleukin-10 (IL-10) protein levels were quantified in the supernatants of PBMC cultures (2 × 106 cells per well; 250 μL) in vitro for 20 h with challenging type 2 PRRSV (0.01 of MOI) or PHA (10 μg/mL) by using a commercial ELISA (Pig Interleukin-10 ELISA kit; Cusabio Biotech, Wuhan, China) according to the manufacturer’s instructions. Detection limits for IL-10 were 1.5 pg/mL.
Macroscopic lung lesion scores
The total extent of macroscopic lung lesions was estimated and calculated as previously described (26). The frequency distribution of the lung lesion scores for each lung lobe was calculated by treatment (16,26).
Morphometric analysis
For the morphometric analysis of pulmonary histopathological changes, 3 lung sections were taken from ventromedial part of the right and left caudal lobe, and right middle lobe. Lung sections were examined blindly and given an estimated severity score for the measures of pulmonary pathology (i.e., atelectasia, epithelial necrosis, hemorrhage, airway plugging, epithelial hyperplasia, interstitial change, and leukocyte infiltration) (26). Each individual measure was given a score ranging from 0 (no lesion visible) to 3 (severe lesions). These scores were added to obtain an overall score for each section.
In-situ hybridization and immunohistochemistry
In-situ hybridization (ISH) for M. hyopneumoniae was done as previously described (27). Immunohistochemistry (IHC) for PRRSV was done as previously described (28). The results of ISH and IHC were analyzed morphometrically as previously described (29,30).
Statistical analysis
Continuous data (rectal body temperatures, ADWG, quantified M. hyopneumoniae DNA and PRRSV RNA, M. hyopneumoniae and PRRSV serology, M. hyopneumoniae- and PRRSV-specific IFN-γ-SC, PRRSV-specific IL-10, macroscopic lung lesion scores, and PRRSV antigen scores) were analyzed using a repeated measures analysis of variance (ANOVA). If the repeated measures ANOVA showed a significant effect, Tukey’s multiple comparison test was done at each time point. Discrete data (clinical respiratory scores, mortality rate, microscopic lung lesion scores, and M. hyopneumoniae DNA scores) were analyzed using the Mann-Whitney test. The correlation between the ADWG and lung lesions was assessed by Spearman’s correlation. A value of P < 0.05 was considered significant.
Results
Clinical evaluation
Pigs in the Zoetis-Vac and BI-Vac groups remained normal throughout the study, as measured by their respiratory scores and rectal temperatures, whereas moderate to severe respiratory signs were observed in the positive controls (Figures 1A and B). The mean rectal temperature (ranging from 39.8°C to 40.2°C) was significantly higher (P < 0.05) in the positive control groups than in the Zoetis-Vac and BI-Vac groups at 3, 4, 5, 6, and 7 dpc (Figure 1B). The mean respiratory scores were significantly higher (P < 0.05) in the positive control group than in the Zoetis-Vac and BI-Vac groups from 14 to 56 dpc, and at 70, 91, and 98 dpc. The overall mortality rate was significantly lower (P < 0.05) for the vaccinated challenged pigs (0%, 0/20 in the Zoetis-Vac group; 5%, 1/20 in the BI-Vac group) than the positive control group (20%; 2/10) (Table I). Diagnostic results indicated that mortality was primarily related to severe pneumonia.
Figure 1.
Mean values of the clinical sign scores (A) and the rectal body temperature (B) in the different groups: Zoetis-Vac (□), BI-Vac (○), positive control (△), and negative control (◇) groups.
a,bSignificant (P < 0.05) difference between groups.
Table I.
Average daily weight gain (ADWG, g/pig per day), proportion of viremic pig and nasal shedder, pathology, in-situ hybridization of Mycoplasma hyopneumoniae (Mhp) and immunohistochemistry of porcine reproductive and respiratory syndrome virus (PRRSV) among 4 groups at different days post challenge (dpc)
| Zoetis-Vac* | BI-Vac† | Positive control | Negative control | |
|---|---|---|---|---|
| Number of pigs | 20 | 20 | 10 | 10 |
| Number of dead pigs | 0 | 1 | 2 | 0 |
| ADWG (wk old) | ||||
| 3–7 | 328 ± 28 | 324 ± 24 | 327 ± 19 | 330 ± 18 |
| 7–10 | 613 ± 26 | 601 ± 30 | 603 ± 28 | 615 ± 26 |
| 10–16 | 806 ± 34a | 786 ± 33a | 729 ± 31b | 802 ± 35a |
| 16–25 | 746 ± 30a | 698 ± 25b | 623 ± 27c | 750 ± 31a |
| 3–25 | 669 ± 25a | 640 ± 23b | 595 ± 20c | 670 ± 24a |
| Mhp nasal shedders (dpc) | ||||
| 14 | 11/20a | 13/20a | 8/10a | 0/10 |
| 28 | 11/20a | 12/19a | 9/9b | 0/10 |
| 63 | 9/20a | 11/19a | 9/9b | 0/10 |
| 91 | 7/20a | 8/19a | 6/8a | 0/10 |
| 126 | 6/20a | 7/19a | 5/8a | 0/10 |
| PRRSV viremic pigs (dpc) | ||||
| 14 | 11/20a | 12/20a | 10/10b | 0/10 |
| 28 | 6/20a | 6/19a | 9/9b | 0/10 |
| 63 | 2/20a | 1/19a | 3/9a | 0/10 |
| 91 | 1/20a | 1/19a | 1/8a | 0/10 |
| 126 | 0/20a | 0/19a | 1/8a | 0/10 |
| PRRSV nasal shedders (dpc) | ||||
| 14 | 10/20a | 12/20a | 10/10b | 0/10 |
| 28 | 6/20a | 7/19a | 8/9b | 0/10 |
| 63 | 1/20a | 1/19a | 3/9a | 0/10 |
| 91 | 1/20a | 1/19a | 1/8a | 0/10 |
| 126 | 0/20a | 0/19a | 0/8a | 0/10 |
| Macroscopic lung lesion score | 2.17 ± 0.63a | 3.48 ± 0.65b | 10.75 ± 5.83c | 0.53 ± 0.38d |
| Microscopic lung lesion score | 2.21 ± 0.34a | 3.10 ± 0.36b | 5.43 ± 0.45c | 0.87 ± 0.35d |
| Mhp DNA score | 0.40 ± 0.51a | 0.55 ± 0.51a | 1.50 ± 0.52b | 0 |
| PRRSV antigen score | 1.95 ± 3.14 | 2.05 ± 3.25 | 2.70 ± 3.02 | 0 |
Pigs that had received RespiSure-One bacterin at 7 days old and Fostera PRRS vaccine at 21 days old followed by a dual challenge at 49 days old.
Pigs that had received Ingelvac MycoFLEX bacterin and Ingelvac PRRS vaccine at 21 days old followed by a dual challenge at 49 days old.
Different letters (a, b, and c) indicate significant (P < 0.05) difference among groups.
Growth performance
No significant difference in the ADWG was observed between Zoetis-Vac and BI-Vac group during weeks 3 to 10. However, during weeks 10 to 16, the ADWG of Zoetis-Vac group was significantly higher (P < 0.05) than that of BI-Vac group. The overall growth performance (from 3 to 25 wk of age) in pigs from the Zoetis-Vac group was significantly higher (P < 0.05) than that of pigs from the BI-Vac group (Table I). The other significant results are summarized in Table I.
Quantification of M. hyopneumoniae DNA in nasal swabs
Prevalence rates of M. hyopneumoniae positive pigs are summarized in Table I. At the time of the challenges, no genomic copies of M. hyopneumoniae were detected in any of the nasal samples from any of the 4 groups. Pigs in the Zoetis-Vac and BI-Vac groups had a significantly lower (P < 0.05) number of genomic copies of M. hyopneumoniae in their nasal swabs than the positive controls throughout the experimental period (Figure 2). Pigs in the Zoetis-Vac group had a significantly lower (P < 0.05) number of genomic copies of M. hyopneumoniae in their nasal swabs than pigs in the BI-Vac group from 14 to 63 dpc. No genomic copies of M. hyopneumoniae were detected in any of the nasal samples from negative controls throughout the experimental period.
Figure 2.
Mean values of the genomic copy numbers of Mycoplasma hyopneumoniae (Mhp) DNA in the nasal swabs in the different groups: Zoetis-Vac (□), BI-Vac (○), and positive control (△) groups.
a,b,cSignificant (P < 0.05) difference between groups.
Quantification of PRRSV RNA in sera and nasal swabs
Prevalence rates of PRRSV positive pigs are summarized in Table I. At the time of the challenges, no genomic copies of PRRSV were detected in any of the serum samples or nasal swabs from any of the 4 groups. Pigs in the Zoetis-Vac and BI-Vac groups had a significantly lower (P < 0.05) number of genomic copies of PRRSV in their sera and nasal swabs than pigs in positive controls at 14 and 28 dpc (Figure 3). No genomic copies of PRRSV were detected in any of the serum samples or nasal swabs from the negative controls throughout the experimental period.
Figure 3.
Mean values of the genomic copy numbers of PRRSV RNA in the serum samples (A) and nasal swabs (B) in the different groups: Zoetis-Vac (□), BI-Vac (○), and positive control (△) groups.
a,b,cSignificant (P < 0.05) difference between groups.
Immunological responses of M. hyopneumoniae
Pigs in the Zoetis-Vac group had significantly higher (P < 0.05) anti-M. hyopneumoniae antibody levels (Figure 4) and numbers of M. hyopneumoniae-specific IFN-γ-SC (Figure 5) at various dpc compared to pigs in the BI-Vac and negative control groups. The other significant results are summarized in Figure 4. No anti-M. hyopneumoniae antibodies or M. hyopneumoniae-specific IFN-γ-SC were detected in the negative controls.
Figure 4.
Immunological responses against Mycoplasma hyopneumoniae (Mhp). Mean values of the anti-M. hyopneumoniae antibody levels. Mean number of M. hyopneumoniae-specific interferon-γ secreting cells (IFN-γ-SC) in the different groups: Zoetis-Vac (□), BI-Vac (○), and positive control (△) groups.
a,b,cSignificant (P < 0.05) difference between groups.
Figure 5.
Immunological responses against Mycoplasma hyopneumoniae (Mhp). Mean number of M. hyopneumoniae-specific interferon-γ secreting cells (IFN-γ-SC) in the different groups: Zoetis-Vac (■), BI-Vac (
), and positive control (□) groups.
a,b,cSignificant (P < 0.05) difference between groups.
Immunological responses of PRRSV
Pigs in the Zoetis-Vac and BI-Vac groups had significantly higher (P < 0.05) anti-PRRSV antibody levels from 0 to 28 dpc compared to positive control group (Figure 6A). The PRRSV-specific NA was detected in pigs in the Zoetis-Vac and BI-Vac groups at 91 and 126 dpc only (Figure 6B). Pigs in the Zoetis-Vac and BI-Vac groups had significantly higher (P < 0.05) numbers of PRRSV-specific IFN-γ-SC from 0 to 28 dpc compared to positive control group (Figure 7). No anti-PRRSV antibodies, PRRSV-specific NA, or PRRSV-specific IFN-γ-SC were detected in the negative controls.
Figure 6.
Immunological responses against porcine reproductive and respiratory syndrome virus (PRRSV). A — Mean values of the anti-PRRSV antibody levels. B — Mean values of PRRSV-specific neutralizing antibodies (NA). Zoetis-Vac (□), BI-Vac (○), and positive control (△) groups.
a,b,cSignificant (P < 0.05) difference between groups.
Figure 7.
Immunological responses against porcine reproductive and respiratory syndrome virus (PRRSV). Mean number of PRRSV-specific interferon-γ secreting cells (IFN-γ-SC) in the different groups: Zoetis-Vac (■), BI-Vac (
), and positive control (□) groups.
a,b,cSignificant (P < 0.05) difference between groups.
The PRRSV-specific IL-10
After stimulation with challenging type 2 PRRSV, IL-10 gradually increased until 0 dpc and thereafter it decreased until 63 dpc in pigs in the Zoetis-Vac and BI-Vac groups (Figure 8). In positive controls, IL-10 had gradually increased at 28 dpc, after which it decreased until 63 dpc. Pigs in the Zoetis-Vac group had significantly lower (P < 0.05) levels of IL-10 than pigs in the BI-Vac group at 0 dpc. Pigs in the Zoetis-Vac and BI-Vac groups had significantly lower (P < 0.05) levels of IL-10 than pigs in the positive group at 28 dpc. Interleukin-10 was not detected in negative controls.
Figure 8.
Level of challenging type 2 porcine reproductive and respiratory syndrome virus (PRRSV)-specific interleukin (IL)-10 in the different groups; Zoetis-Vac (□), BI-Vac (○), and positive control (△) groups.
a,b,cSignificant (P < 0.05) difference between groups.
Lung lesion scores
Pigs in the Zoetis-Vac group had significantly lower (P < 0.05) scores for macroscopic and microscopic pulmonary lesions than pigs in the BI-Vac group at 126 dpc. The other significant results of scores for pulmonary lesions are summarized in Table I. Additionally, a negative correlation was found between ADWG and macroscopic lung lesion (r = −0.578, P < 0.01), and between ADWG and microscopic lung lesion (r = −0.538, P < 0.01).
In-situ hybridization of M. hyopneumoniae
Pigs in the Zoetis-Vac and BI-Vac groups had significantly (P < 0.05) lower amounts of M. hyopneumoniae DNA in their lungs than positive controls (Table I).
Immunohistochemistry for PRRSV
There was no significantly different number of PRRSV-positive cells per unit in the lungs from pigs in the Zoetis-Vac, BI-Vac, or positive control groups (Table I).
Discussion
The present study demonstrated that the single-dose vaccination regimen for M. hyopneumoniae bacterin and PRRS modified live vaccine is efficacious for controlling coinfection with M. hyopneumoniae and PRRSV from wean to finish based on clinical, microbiological, immunological, and pathological evaluation. Vaccination of pigs with M. hyopneumoniae and PRRSV is able to reduce the levels of mycoplasmal nasal shedding, PRRSV viremia, and severity of lung lesions compared to unvaccinated challenged pigs. In contrast with earlier findings that vaccination with the PRRSV vaccine reduced the efficacy of the M. hyopneumoniae bacterin (31), sequential or concurrent single-dose vaccination with M. hyopneumoniae and PRRSV could not reduce the efficacy of the M. hyopneumoniae bacterin or the PRRSV vaccine in this study. Our results agree with previous findings in which no negative influence of the PRRSV vaccination on M. hyopneumoniae vaccine efficacy was observed (32,33). Interestingly, high levels of IL-10 were detected in pigs during the first 4 to 5 wk following PRRSV vaccination. Because IL-10 is a known potent immunosuppressive cytokine (34), induction of IL-10 following PRRSV vaccination may cause interference of the mycoplasmal vaccine’s efficacy. However, M. hyopneumoniae-specific IFN-γ-SCs gradually increased at the same time as the gradual increase of IL-10, indicating that levels of IL-10 following PRRSV vaccination are not enough to interfere with immune responses induced by the mycoplasmal vaccine.
Selection of an appropriate challenge virus is critical when comparing 2 PRRSV vaccines, which are only 91.7% homologous to each other. The Ingelvac PRRS MLV vaccine virus is from lineage 5 and the Fostera PRRS vaccine virus is from lineage 8. A challenge strain was chosen that was not closely related to either vaccine virus and had a similar level of homology to the 2 vaccine viruses, based on open reading frame 5 (ORF5) nucleotide sequences. Strain SNUVR090851 is from lineage 1 with homologies of 87.2% and 85.9% to Fostera PRRS and Ingelvac PRRS MLV, respectively. However, our results should be interpreted carefully because only ORF5 of the genome is used for the comparison. Therefore, additional studies are needed to confirm heterogenicity using full genome of challenge and vaccine viruses.
Pathological observation is also important parameter to evaluate the mycoplasmal and PRRSV vaccine because a reduction in M. hyopneumoniae- and PRRSV-induced lesions is correlated with improved weight gain (35–38). In the present study, coinfection with M. hyopneumoniae and PRRSV induces severe lung lesions, which are similar to typical PRDC. The most striking and consistent microscopic lesions were severe interstitial pneumonia with some degree of peribronchial and peribronchiolar fibrosis and lymphoid tissue hyperplasia. A reduction in lung lesions was correlated with improved weight gain, as previous studies have shown (35–38).
Comparison of 2 vaccines is more practical and may be reflected field conditions because M. hyopneumoniae and PRRSV are 2 major contributors to PRDC (10). The 2 commercial vaccines used in this study were shown to be efficacious in controlling coinfection with M. hyopneumoniae and PRRSV based on clinical, microbiological, immunological, and pathological evaluation under experimental conditions.
Acknowledgments
This research was supported by Zoetis, 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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