Abstract
The objective of this study was to assess the minimum dose, antigen content, and immunization duration of a trivalent vaccine containing inactivated Haemophilus parasuis serovars 4, 5, and 12 and the Montanide GEL 01 PR adjuvant in piglets and pregnant sows. Our results demonstrated that the minimum vaccine dose was 2 mL per pig and the optimal antigen content 2.0 × 109, 1.0 × 109, and 1.0 × 109 colony-forming units/mL of serovars 4, 5, and 12, respectively. The vaccine provided effective protection 14 d after the 2nd vaccination, and the period of immune protection was 180 d (6 mo) after the 2nd vaccination. Maternal antibodies provided early protection for the piglets, and vaccinating the sows before farrowing helped to control disease and protected the piglets during lactation; the piglets were protected during the finishing period by being vaccinated during lactation. Our findings provide a basis for developing a commercial trivalent vaccine of inactivated H. parasuis serovars 4, 5, and 12 against Glässer’s disease.
Résumé
L’objectif de la présente étude était d’évaluer la dose minimale, le contenu en antigène, et la durée d’immunisation d’un vaccin trivalent contenant les sérovars 4, 5, et 12 d’Haemophilus parasuis et l’adjuvant Montanide GEL 01 PR chez des porcelets et des truies gestantes. Nos résultats ont démontré que la dose minimale de vaccin était de 2 mL par porc et le contenu optimal en antigène était de 2,0 × 109, 1,0 × 109, and 1,0 × 109 unités formatrices de colonie/mL pour les sérovars 4, 5, et 12, respectivement. Le vaccin a fourni une protection efficace 14 j suite à la deuxième vaccination, et la période de protection immunitaire était de 180 j (6 mois) après la deuxième vaccination. Les anticorps maternels ont fourni une protection précoce aux porcelets, et la vaccination des truies avant la mise-bas aidait à maitriser la maladie et a protégé les porcelets durant la lactation; les porcelets étaient protégés durant la période de finition en étant vaccinés durant la lactation. Nos trouvailles fournissent des éléments de base pour développer un vaccin trivalent commercial contre la maladie de Glässer avec des souches inactivées d’H. parasuis sérovars 4, 5, et 12.
(Traduit par Docteur Serge Messier)
Introduction
Haemophilus parasuis, which belongs to the Pasteurellaceae family, can cause severe infections of the upper respiratory tract, polyserositis, meningitis, and arthritis (Glässer’s disease) in pigs (1). To date, H. parasuis has mainly caused significant losses among piglets 4 to 12 wk old (2,3). Fifteen serovars of H. parasuis, which range from highly virulent to nonvirulent, have been described by immunodiffusion tests and multiplex polymerase chain reaction (PCR). Sequence variations within the capsule loci can distinguish all 15 serovars except serovars 5 and 12 (1,4). Moreover, the pathogenicity and prevalence of different H. parasuis serovars may vary among regions and over time within a region (5–8).
Vaccination is generally considered to be the most effective means of controlling Glässer’s disease, which normally has high mortality and morbidity rates. Inactivated H. parasuis bacterin, which is used worldwide, can elicit efficient protection against challenge with a homologous serovar. However, disease control is limited because of the lack of effective vaccines against a broad spectrum of strains (9–11). Recently, subunit vaccines that comprise newly identified protective antigens, such as recombinant transferrin-binding protein B (TbpB), outer membrane protein (OMP) formulations enriched with TbpB, 4 OMPs (OMP2, D15, PalA, and HPS-06257), or transferrin-binding protein A, have been proven to provide partial protection against H. parasuis challenge (9,10,12). All types of vaccines usually provide protection against challenge with homologous serovars, but few studies have reported cross-protection (2,12,13). To date, vaccines against Glässer’s disease containing inactivated H. parasuis serovars 4 and 5, serovar 5, and serovars 1 and 6 are the primary commercial vaccines in China, the United States, and Spain, respectively. These vaccines play an important role in preventing and controlling Glässer’s disease (14).
In a previous study we screened the strongly immunogenic serovars 4, 5, and 12 as candidate vaccine strains and compared the effectiveness of vaccines containing inactivated H. parasuis serovars 4, 5, and 12 when administered with a variety of adjuvants [traditional mineral oil, aluminum hydroxide, Montanide GEL 01 PR (a new adjuvant based on the dispersion of a high-molecular-weight polyacrylic polymer in water; SEPPIC, Shanghai, China), Montanide IMS 1313N VG, and Montanide ISA 760 VG]. We concluded that the Montanide GEL 01 PR should be used as a candidate adjuvant, deserving further study because a vaccine administered with this adjuvant was safe, generated high concentrations of antibodies, and exhibited 100% protection (15). The objectives of the present study were to assess the minimum dose, antigen content, and immunization duration of a trivalent vaccine containing inactivated H. parasuis serovars 4, 5, and 12 administered to pigs for the first time and to provide a basis for developing a commercial trivalent vaccine with these inactivated serovars to protect against Glässer’s disease.
Materials and methods
Animals
Female Landrace × Large White piglets aged 3 to 4 wk old and Landrace × Large White sows that were 8 to 9 wk pregnant were purchased from ShengPing Co., Ltd, Luoyang, China. A single nasal swab obtained from each pig was shown by PCR to be negative for H. parasuis as described previously (15). The pigs were determined to be serologically negative by a microagglutination test (MAT) developed in our laboratory. Briefly, 50 μL of sterile PBS was added to 96-well plates, along with 50 μL of test serum diluted 2-fold with sterile PBS. Into each well was mixed 50 μL of inactivated H. parasuis whole-cell antigen. The mixture was incubated at 37°C for 7 h and then at 4°C for 12 to 24 h. Titers ≥ 1:8 and ≤ 1:4 were considered to be positive and negative, respectively.
All the pigs were housed in isolation in pens with a concrete floor. Feed and water were provided ad libitum throughout the study. All the animal procedures were conducted in compliance with the guidelines of the Animal Care and Use Committee of Henan University of Science and Technology, Luoyang, China.
Isolates of H. parasuis
Isolates of H. parasuis, serovars 4, 5, and 12, that were screened by our laboratory (16) were used for vaccine production. The bacteria were cultured on tryptic soy agar (TSA) supplemented with nicotinamide adenine dinucleotide (NAD), 10 μg/mL, and 5% fetal calf serum and incubated at 37°C for 48 h. The organisms were then washed off the TSA plates with sterile 15% skimmed milk powder and stored at −80°C until used.
Vaccine formulations and vaccination schedule
Four vaccines (F1 to F4) were compared. Briefly, H. parasuis serovars 4, 5, and 12 were enumerated by plate counts. Then the bacterial cultures were inactivated by incubation with 0.3% formaldehyde solution at 37°C for 48 h and pelleted by high-speed centrifugation. The pellets were resuspended in sterile phosphate-buffered saline (PBS) as whole-cell antigens and subsequently formulated with Montanide GEL 01 PR adjuvant (kindly provided by SEPPIC) at an 85:15 ratio (H. parasuis:adjuvant) in accordance with the manufacturer’s instructions. The vaccines contained serovars 4, 5, and 12 at the following antigen concentrations: F1, 2.0 × 109, 1.0 × 109, and 1.0 × 109 colony-forming units (CFU)/mL, respectively; F2, 1.0 × 109, 5.0 × 108, and 5.0 × 108 CFU/mL, respectively; F3, 5.0 × 108, 2.5 × 108, and 2.5 × 108 CFU/mL, respectively; and F4, 2.5 × 108, 1.3 × 108, and 1.3 × 108 CFU/mL, respectively. The physical properties and sterility of the vaccines were described by the Veterinary Biological Products Procedures of the People’s Republic of China.
All the vaccines were administered intramuscularly twice (21 d apart) in the neck. The day of the 1st vaccination was designated as d 0. At 14 d after the 2nd vaccination all the piglets were challenged intraperitoneally with 9.0 × 109 CFU (15) of H. parasuis serovar 4 (approximately 3 mL), 5.2 × 109 CFU (15) of H. parasuis serovar 5 (approximately 2 mL), and 3.5 × 109 CFU (15) of H. parasuis serovar 12 (approximately 1.5 mL); the bacteria had been cultured in trypticase soy broth supplemented with NAD, 10 μg/mL, and 5% fetal calf serum for 16 h. Control pigs were injected with 2 mL of sterile PBS on the same day that the other groups were vaccinated. Blood samples from the piglets were collected by puncture of the jugular vein.
Minimum vaccine dose for piglets
Sixty piglets were randomly assigned to 12 groups (A1 to A12) of 5 animals each. The piglets in groups A1 to A3, A4 to A6, and A7 to A9 were vaccinated with 2, 1, and 0.5 mL, respectively, of the F1 vaccine. The piglets in groups A10 to A12 served as controls. Groups A1, A4, A7, and A10 were challenged with H. parasuis serovar 4; groups A2, A5, A8, and A11 were challenged with serovar 5; and groups A3, A6, A9, and A12 were challenged with serovar 12.
Blood samples from the piglets were collected before challenge (on d 35). The serum was stored at −80°C until the MAT analysis. The experiment was terminated 15 d after challenge; survivors were killed by intravenous administration of sodium pentobarbital. Piglets found moribund during the experiment were humanely killed in the same manner. All of the animals underwent necropsy; gross lesions were recorded (special attention was paid to the pleural, pericardial, and peritoneal cavities, the lungs, and the central nervous system), and samples were obtained for bacterial isolation.
Minimum vaccine dose for pregnant sows
Twenty pregnant sows were randomly assigned to 4 groups (B1 to B4) of 5 animals each. The sows in groups B1, B2, and B3 were vaccinated with 2, 1, and 0.5 mL, respectively, of the F1 vaccine. The sows in groups B4 served as controls.
Blood samples from piglets born to the pregnant sows were collected at 1, 4, 7, 10, 15, 20, 25, 30, 35, 40, 45, and 50 d of age. Serum antibody titers were analyzed to assess dynamic changes in the H. parasuis antibody levels in these piglets. Fifteen piglets 21 to 22 d old were selected from each group of sows. Then 15 piglets were randomly assigned to 3 groups of 5 piglets each, and immediately challenged with H. parasuis serovars 4, 5, and 12, respectively. The procedures after challenge were as described.
Effect of vaccine antigen content on piglets
Seventy-five piglets were randomly assigned to 15 groups (C1 to C15) of 5 animals each. The piglets in groups C1 to C3, C4 to C6, C7 to C9, and C10 to C12 were vaccinated with 2 mL of the F1, F2, F3, and F4 vaccines, respectively. The piglets in groups C13 to C15 served as controls. Groups C1, C4, C7, C10, and C13 were challenged with serovar 4; groups C2, C5, C8, C11, and C14 were challenged with serovar 5; and groups C3, C6, C9, C12, and C15 were challenged with serovar 12.
Blood samples from the piglets were collected before challenge (on d 35), and the serum was stored at −80°C until the MAT analysis. The procedures after challenge were as described.
Effect of vaccine antigen content on pregnant sows
Twenty-five pregnant sows were randomly assigned to 4 groups (D1 to D5) of 5 animals each. The sows in groups D1, D2, D3, and D4 were vaccinated with 2 mL of the F1, F2, F3, and F4 vaccines, respectively. The sows in groups D5 served as controls. The procedures on the piglets born to these sows were as described.
Immunization duration in piglets
One hundred and fifty piglets were randomly assigned to 2 groups (E1 and E2) of 75 animals each. The piglets in group E1 were vaccinated with 2 mL of the F1 vaccine. The piglets in group E2 served as controls. Blood samples were collected on days 0 and 21 after the 1st vaccination and on days 7, 14, 28, 60, 90, 120, 150, 180, 210, and 240 after the 2nd vaccination. Serum titers of H. parasuis antibody were analyzed to assess dynamic changes in the levels.
Fifteen piglets were selected from each group at 14, 90, 180, and 210 d after the 2nd vaccination and then randomly assigned to 3 groups of 5 piglets each. The piglets were immediately challenged with H. parasuis serovars 4, 5, and 12, respectively. The procedures after challenge were as described.
Immunization duration in pregnant sows
Twelve pregnant sows were randomly assigned to 2 groups (Z1 and Z2) of 6 animals each. The sows in group Z1 were vaccinated with 2 mL of the F1 vaccine. Group Z2 served as controls. Blood samples from the sows were collected on days 0 and 21 after the 1st vaccination and on days 7, 14, 28, 60, 90, 120, 150, 180, 210, and 240 after the 2nd vaccination. Serum titers of H. parasuis antibody were analyzed to assess dynamic changes in the levels.
Blood samples from piglets born to the pregnant sows were collected at 1, 4, 7, 10, 15, 20, 25, 30, 35, 40, 45, and 50 d of age. Serum titers of H. parasuis antibody were analyzed to assess dynamic changes in the levels. Fifteen piglets aged 21 to 22 d, 34 to 36 d, and 56 to 58 d were selected from each group and then randomly assigned to 3 groups of 5 piglets each and immediately challenged with H. parasuis serovars 4, 5 and 12, respectively. The procedures after challenge were as described.
Statistical analysis
Statistical analysis was done by means of 1-way analysis of variance. A P-value < 0.05 was considered to be significant. The analysis was conducted with the use of SPSS Statistics for Windows, version 17.0 (SPSS, Chicago, Illinois, USA).
Results
As Table I shows for the vaccine-dose experiments, among the piglets the mean titers of antibody to serovars 4, 5, and 12 were significantly higher (P < 0.05) in groups A1 to A3 (vaccinated with 2 mL of the F1 vaccine) than in groups A4 to A6 and groups A7 to A9 (vaccinated with 1 mL and 0.5 mL of the F1 vaccine, respectively). The mean titers did not differ significantly between groups A4 to A6 and groups A7 to A9. No H. parasuis antibodies were observed in groups A10 to A12, the unvaccinated control piglets. After challenge with H. parasuis serovars 4, 5, and 12, all the control piglets had signs of Glässer’s disease, including anorexia, psychiatric disorders, arthritis, convulsions, serofibrinous exudates, and polyserositis of varying severity within the pericardium, thoracic cavity, and enterocelia; these piglets began to die 6 h after challenge, and all had died by 72 h. Vaccination with 0.5 mL of the F1 vaccine provided 60% protection against challenge, whereas vaccination with 1 mL provided 100% protection except against serovar 5 (80%), and vaccination with 2 mL provided 100% protection. The organism was isolated from tissues and organs of all of the dead experimental piglets, which had exhibited symptoms and pathological changes of Glässer’s disease in the upper respiratory tract similar to those in the controls. None of the surviving piglets exhibited similar symptoms or pathological changes.
Table I.
Protective efficacy of the F1 vaccinea against serovars 4, 5, and 12 of Haemophilus parasuis and mean titers of antibody before challenge in piglets that had received various doses of the vaccine
| Piglet group | Number of piglets | Vaccine dose (mL) | Protective efficacy (and mean antibody titerb) | ||
|---|---|---|---|---|---|
|
| |||||
| Serovar 4 | Serovar 5 | Serovar 12 | |||
| A7 to A9 | 15 | 0.5 | 60% (1:12) | 60% (1:11) | 60% (1:12) |
| A4 to A6 | 15 | 1 | 100% (1:18) | 80% (1:16) | 100% (1:18) |
| A1 to A3 | 15 | 2 | 100% (1:28) | 100% (1:37) | 100% (1:37) |
| Controlsc (A10 to A12) | 15 | 2 | 0% (—) | 0% (—) | 0% (—) |
The vaccine, given as 2 intramuscular doses 21 d apart, contained serovars 4, 5, and 12 of H. parasuis at antigen contents of 2.0 × 109, 1.0 × 109, and 1.0 × 109 colony-forming units (CFU)/mL, respectively. The piglets were challenged intraperitoneally with H. parasuis 14 d after the second vaccination.
The mean antibody titers in groups A1 to A3 were significantly higher (P < 0.05) than those in the other experimental groups.
The piglets in the control group were injected with 2 mL of sterile phosphate-buffered saline the same days as the other groups were vaccinated. The dash indicates that no H. parasuis antibodies were detected.
Dynamic changes in the titers of maternal antibodies against H. parasuis in the piglets born to the pregnant sows are shown in Table II for the vaccine-dose experiments. The mean titers in the piglets born to the B1 and B2 groups (vaccinated with 2 mL and 1 mL of the F1 vaccine, respectively) were highest at day 20 and gradually decreased thereafter. However, the titers decreased faster in the B2 group than in the B1 group. The mean titer in the piglets born to the B3 group (vaccinated with 0.5 mL of the F1 vaccine) was highest at day 15 and then gradually decreased; however, the titers in these piglets were the lowest (≤ 1:16) and were maintained for a short time. The mean titer in the piglets born to the B1 group was not significantly different from that of the piglets born to the B2 group but was significantly higher (P < 0.05) than that of the piglets born to the B3 group. The mean titer in the piglets born to the B2 group was not significantly different from that of the B3 group. No maternal antibodies were observed in the controls (group B4). Among the 15 piglets 21 to 22 d old that were selected from each group and challenged with H. parasuis serovars 4, 5, and 12 the protective efficacy of the vaccine was the same as for groups A1 to A3, A4 to A6, A7 to A9, and A10 to A12.
Table II.
Mean titers of maternal antibody to H. parasuis serovars 4, 5, and 12 in piglets born to pregnant sows that had received various doses of the F1 vaccine
| Piglet age (days) | Vaccine dose, mL (and sow group); mean antibody titera | |||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
|
| ||||||||||||
| Serovar 4 | Serovar 5 | Serovar 12 | ||||||||||
|
|
|
|
||||||||||
| 2 (B1) | 1 (B2) | 0.5 (B3) | 0 (B4) | 2 (B1) | 1 (B2) | 0.5 (B3) | 0 (B4) | 2 (B1) | 1 (B2) | 0.5 (B3) | 0 (B4) | |
| 1 | — | — | — | — | — | — | — | — | — | — | — | — |
| 4 | 1:4 | 1:4 | 1:2 | — | 1:4 | 1:4 | 1:2 | — | 1:4 | 1:4 | 1:2 | — |
| 7 | 1:8 | 1:8 | 1:4 | — | 1:8 | 1:4 | 1:8 | — | 1:4 | 1:8 | 1:8 | — |
| 10 | 1:16 | 1:16 | 1:8 | — | 1:16 | 1:16 | 1:8 | — | 1:16 | 1:16 | 1:8 | — |
| 15 | 1:32 | 1:32 | 1:8 | — | 1:32 | 1:32 | 1:16 | — | 1:32 | 1:32 | 1:16 | — |
| 20 | 1:32 | 1:32 | 1:8 | — | 1:64 | 1:64 | 1:8 | — | 1:32 | 1:32 | 1:8 | — |
| 25 | 1:32 | 1:32 | 1:4 | — | 1:32 | 1:16 | 1:8 | — | 1:16 | 1:16 | 1:8 | — |
| 30 | 1:16 | 1:16 | 1:2 | — | 1:16 | 1:16 | 1:4 | — | 1:16 | 1:16 | 1:4 | — |
| 35 | 1:16 | 1:16 | — | — | 1:16 | 1:8 | 1:2 | — | 1:8 | 1:8 | 1:2 | — |
| 40 | 1:8 | 1:8 | — | — | 1:8 | 1:4 | — | — | 1:4 | 1:4 | — | — |
| 45 | 1:4 | 1:2 | — | — | 1:2 | — | — | — | 1:2 | 1:2 | — | — |
| 50 | — | — | — | — | — | — | — | — | — | — | — | — |
Antibody titers ≥ 1:8 and ≤ 1:4 were deemed to be positive and negative, respectively. The mean titer in the piglets born to the B1 group was not significantly different from that of the piglets born to the B2 group but was significantly higher (P < 0.05) than that of the piglets born to the B3 group. The mean titers in the piglets born to the B2 and B3 groups did not differ significantly. The dash indicates that no H. parasuis antibodies were detected.
In the antigen-content experiments the mean titers of antibody to serovars 4, 5, and 12 in the piglets (Table III) were significantly higher (P < 0.05) in groups C1 to C3 (vaccinated with the F1 vaccine) than in groups C4 to C6 (vaccinated with the F2 vaccine). The antibody titers in those groups were significantly higher (P < 0.05) than the titers in groups C7 to C9 and C10 to C12 (vaccinated with the F3 and F4 vaccines, respectively). The mean titers did not differ significantly between groups C7 to C9 and groups C10 to C12. No antibodies against H. parasuis serovars 4, 5, and 12 were observed in the control piglets (groups C13 to C15), all of which showed signs of Glässer’s disease after challenge and died by 72 h after challenge. The F1 vaccine provided 100% protection against challenge with H. parasuis serovars 4, 5, and 12, and the F2 vaccine provided 100% protection against challenge with H. parasuis serovars 5 and 12 but only 80% protection against serovar 4, whereas the F3 and F4 vaccines provided little protection against the 3 serovars. The organism was isolated from tissues and organs of all of the dead experimental piglets, which exhibited symptoms and pathological changes of Glässer’s disease in the upper respiratory tract similar to those in the controls. None of the surviving piglets exhibited similar symptoms or pathological changes.
Table III.
Protective efficacy of the F1, F2, F3, and F4 vaccinesa against serovars 4, 5, and 12 of H. parasuis and mean antibody titers before challenge in piglets that had received 2 mL of vaccine
| Vaccine type (and piglet group) | Number of piglets | Protective efficacy (and mean antibody titerb) | ||
|---|---|---|---|---|
|
| ||||
| Serovar 4 | Serovar 5 | Serovar 12 | ||
| F1 (C1 to C3) | 15 | 100% (1:28) | 100% (1:28) | 100% (1:37) |
| F2 (C4 to C6) | 15 | 80% (1:16) | 100% (1:18) | 100% (1:18) |
| F3 (C7 to C9) | 15 | 40% (1:12) | 60% (1:11) | 40% (1:11) |
| F4 (C10 to C12) | 15 | 0% (1:8) | 0% (1:8) | 20% (1:8) |
| Controlsc (C13 to C15) | 15 | 0% (—) | 0% (—) | 0% (—) |
The vaccines were given as 2 intramuscular doses 21 d apart. They contained serovars 4, 5, and 12 of H. parasuis at the following antigen concentrations: F1, 2.0 × 109, 1.0 × 109, and 1.0 × 109 CFU/mL, respectively; F2, 1.0 × 109, 5.0 × 108, and 5.0 × 108 CFU/mL, respectively; F3, 5.0 × 108, 2.5 × 108, and 2.5 × 108 CFU/mL, respectively; and F4, 2.5 × 108, 1.3 × 108, and 1.3 × 108 CFU/mL, respectively. The piglets were challenged intraperitoneally with H. parasuis 14 d after the 2nd vaccination.
The mean antibody titers in groups C1 to C3 were significantly higher (P < 0.05) than those in the other experimental groups.
The piglets in the control group were injected with 2 mL of sterile phosphate-buffered saline the same days as the other groups were vaccinated. The dash indicates that no H. parasuis antibodies were detected.
Dynamic changes in the maternal antibody titers of the piglets born to pregnant sows in groups D1, D2, and D3 (vaccinated with the F1, F2, and F3 vaccines, respectively) were similar to those of the piglets born to pregnant sows in groups B1, B2, and B3 (Table II), respectively. The piglets in group D4 (vaccinated with the F4 vaccine) had the lowest mean titer (≤ 1:8), which was maintained for only a short time. No maternal antibodies against H. parasuis serovars 4, 5, and 12 were observed in the piglets in the control group. Among the 15 piglets 21 to 22 d old that were selected from each group and challenged with H. parasuis serovars 4, 5, and 12 the protective efficacies of the vaccine for the piglets in the D1 to D4 groups were identical to those in the C1 to C3, C4 to C6, C7 to C9, and C10 to C12 groups (Table III), respectively.
Dynamic changes in the titers of antibody to H. parasuis serovars 4, 5, and 12 in the piglets in the immunization-duration experiment are shown in Table IV. The mean titers gradually increased from the 1st vaccination to day 28 after the 2nd vaccination and then gradually decreased, disappearing by day 210. The piglets were challenged with H. parasuis serovars 4, 5, and 12 at 14, 90, 180, and 210 d after the 2nd vaccination. One piglet challenged with H. parasuis serovar 4 died 10 d after challenge at d 210, whereas all of the other experimental piglets survived; all of the control piglets had signs of Glässer’s disease and died by 72 h after challenge. The organism was isolated from tissues and organs of the dead experimental piglets, which exhibited symptoms and pathological changes of Glässer’s disease in the upper respiratory tract similar to those in the control groups. None of the surviving piglets exhibited similar symptoms and pathological changes.
Table IV.
Dynamic changes in the mean titers of antibody to H. parasuis serovars 4, 5, and 12 in 3 groups of 5 piglets each that received 2 mL of the F1 vaccinea (E1) and 15 controlsb (E2)
| Serovar | Piglet group | Day after first vaccination (0 or 21) or 2nd vaccination; mean antibody titerc | |||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
|
| |||||||||||||
| 0 | 21 | 7 | 14 | 28 | 60 | 90 | 120 | 150 | 180 | 210 | 240 | ||
| 4 | E1 | — | 1:8 | 1:16 | 1:32 | 1:64 | 1:64 | 1:32 | 1:32 | 1:16 | 1:16 | 1:8 | 1:4 |
| 5 | E1 | — | 1:8 | 1:16 | 1:32 | 1:128 | 1:64 | 1:64 | 1:32 | 1:32 | 1:16 | 1:16 | 1:4 |
| 12 | E1 | — | 1:8 | 1:16 | 1:32 | 1:128 | 1:64 | 1:64 | 1:32 | 1:32 | 1:16 | 1:16 | 1:4 |
| E2 | — | — | — | — | — | — | — | — | — | — | — | — | |
The vaccine was given as 2 intramuscular doses 21 d apart.
The controls were injected with 2 mL of sterile phosphate-buffered saline the same days as the other piglets were vaccinated.
Antibody titers ≥ 1:8 and ≤ 1:4 were deemed to be positive and negative, respectively. The dash indicates that no H. parasuis antibodies were detected.
Dynamic changes in the titers of antibody to H. parasuis serovar 4, 5, and 12 in the pregnant sows are shown in Table V. The titers increased more slowly than those in the piglets represented in Table IV from the 1st vaccination to day 14 after the 2nd vaccination, but other changes were similar to those for the piglets. Dynamic changes in the titers of maternal antibody in the piglets born to pregnant sows were identical to those in the B1 group (vaccinated with 2 mL of the F1 vaccine) as shown in Table II. All the piglets aged 21 to 22 d and 60% of those aged 34 to 36 d survived challenge with H. parasuis serovars 4, 5, and 12, whereas all of the piglets aged 56 to 58 d, as well as the control piglets, died after challenge. The organism was isolated from tissues and organs of all of the dead piglets, which had signs of Glässer’s disease. None of the surviving piglets exhibited symptoms and pathological changes of Glässer’s disease in the upper respiratory tract, as occurred in the control group.
Table V.
Dynamic changes in the mean titers of antibody to H. parasuis serovars 4, 5, and 12 in pregnant sows, 6 of which received 2 mL of the F1 vaccinea (Z1) while the other 6 served as controlsb (Z2)
| Serovar | Sow group | Day after first vaccination (0 or 21) or 2nd vaccination; mean antibody titerc | |||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
|
| |||||||||||||
| 0 | 21 | 7 | 14 | 28 | 60 | 90 | 120 | 150 | 180 | 210 | 240 | ||
| 4 | Z1 | — | 1:4 | 1:8 | 1:16 | 1:64 | 1:64 | 1:32 | 1:32 | 1:16 | 1:16 | 1:8 | 1:4 |
| 5 | Z1 | — | 1:4 | 1:8 | 1:16 | 1:128 | 1:64 | 1:64 | 1:32 | 1:32 | 1:16 | 1:16 | 1:4 |
| 12 | Z1 | — | 1:4 | 1:8 | 1:16 | 1:128 | 1:64 | 1:64 | 1:32 | 1:16 | 1:16 | 1:16 | 1:4 |
| Z2 | — | — | — | — | — | — | — | — | — | — | — | — | |
The vaccine was given as 2 intramuscular doses 21 d apart.
The controls were injected with 2 mL of sterile phosphate-buffered saline the same days as the other sows were vaccinated.
Antibody titers ≥ 1:8 and ≤ 1:4 were deemed to be positive and negative, respectively. The dash indicates that no H. parasuis antibodies were detected.
Discussion
Glässer’s disease has high morbidity and mortality rates in pigs in China and some European countries. Its prevention and control are major problems worldwide. To date, because of a lack of knowledge regarding the virulence factors and common protective antigens of H. parasuis, effective vaccines that provide cross-protection against all pathogenic serovars are not available (12). Additionally, no other commercially available vaccine types, such as subunit vaccines and genetically engineered attenuated H. parasuis vaccines, that are effective against pathogenic H. parasuis serovars have been developed. Therefore, the development of commercially available multivalent vaccines containing inactivated field isolates is necessary to protect against Glässer’s disease.
In China, H. parasuis serovars 4 and 5 are the most prevalent, followed by serovars 14, 13, and 12 (5–8). A vaccine containing inactivated H. parasuis serovars 4 and 5 can reduce the severity of symptoms and the risk of death in pigs after challenge with serovars 13 and 14 but fail to protect them from infection with serovar 12 (17). Additionally, to the best of our knowledge, no commercially available vaccines against H. parasuis serovar 12 have been developed. Hence we assessed a trivalent vaccine containing inactivated H. parasuis serovars 4, 5, and 12, administered with the Montanide GEL 01 PR adjuvant, in this study.
It is well-known that H. parasuis mainly infects the upper respiratory tract of pigs. In this study, classic symptoms of Glässer’s disease were observed in the upper respiratory tract of the unvaccinated pigs, whereas the vaccinated pigs were protected at the infection site (upper respiratory tract) even though an intraperitoneal challenge was used.
Our experiments that aimed to determine the minimum vaccine dose for piglets and pregnant sows showed that the concentration of antibodies against H. parasuis serovars 4, 5, and 12 increased with increasing vaccine dose: the antibody titers induced by 2 mL of the F1 vaccine were significantly higher (P < 0.05) than the titers induced in the other groups of vaccinees, and 2 mL of the F1 vaccine provided 100% protection against challenge with H. parasuis serovars 4, 5, and 12. These results revealed that the minimum vaccine dose was 2 mL per pig. The experiments that examined the effect of the antigen content on piglets and pregnant sows showed that the concentration of antibodies against H. parasuis serovars 4, 5, and 12 increased with increasing antigen content: the antibody titers induced by 2 mL of the F1 vaccine were significantly higher (P < 0.05) than the titers induced in the other groups of vaccinees, and 2 mL of the F1 vaccine provided 100% protection against challenge with H. parasuis serovars 4, 5, and 12. These results suggest that the optimal antigen contents of H. parasuis serovars 4, 5, and 12 are 2.0 × 109, 1.0 × 109, and 1.0 × 109 CFU/mL, respectively.
The minimum vaccine dose and optimal antigen content of the trivalent vaccine containing inactivated H. parasuis serovars 4, 5, and 12 were used to assess the immunization duration in piglets and pregnant sows. In these experiments, antibody titers of 1:16 or greater were observed from 14 to 180 d after the 2nd vaccination, and all of the piglets survived challenge with H. parasuis serovars 4, 5, and 12 at 14, 90, and 180 d after the 2nd vaccination. These results indicate that the trivalent vaccine provided effective protection at d 14 after the 2nd vaccination and that the immune protection lasted until 180 d (6 mo) after the 2nd vaccination. Our results also indicate that the 1st vaccination should be done in sows that have been pregnant for 8 to 9 wk, because high antibody concentrations could be detected in the vaccinated pregnant sows during farrowing. Additionally, sows should be given a booster injection 21 d later, because neonatal piglets could receive more maternal antibodies that allow them to survive without resulting disease while they produce an active immune response.
The maternal antibodies that are induced by commercially available bacterin containing H. parasuis serovars 4 and 5 or containing H. parasuis serovars 2, 3, and 5 can provide early protection for piglets (18–20). The results obtained with the piglets born to vaccinated sows in this study support this view. Additionally, we detected maternal antibodies in neonatal piglets and dynamic changes in the antibody titers in vaccinated piglets aged 3 to 9 wk, as well as in pregnant sows. Our findings suggest that the maternal antibody titers were maintained for 6 wk (Table II) and therefore that vaccinating sows before farrowing may help to control the disease and protect the piglets during lactation. However, the susceptibility to H. parasuis infection and disease increased in piglets after weaning because of the disappearance of maternal antibodies and the loss of passive protection. Therefore, it is important that piglets be inoculated with an effective vaccine before the disappearance of maternal antibodies. In this study the maternal antibody concentration was highest in the piglets at 20 d of age and then gradually decreased. Thus, we recommend that the 1st vaccination be done in piglets 3 to 4 wk old and that they be given a booster injection 21 d later, as this vaccination regimen provided protection against H. parasuis serovars 4, 5, and 12 for 180 d. Our results showed that the vaccinated offspring of the vaccinated sows had longer-lasting serologic protection, which would help to prevent H. parasuis colonization.
Our findings provide new, detailed data regarding the vaccine optimization that is needed to guarantee the efficacy of a trivalent vaccine against Glässer’s disease in piglets and pregnant sows containing inactivated H. parasuis serovars 4, 5, and 12, and they provide a basis for developing a commercial vaccine. Meanwhile, the practical significance of our findings is that if H. parasuis infections are endemic in a swine-breeding facility it is necessary to vaccinate the sows before farrowing. The piglets could be protected during the finishing period if they are vaccinated during lactation. Field trials of the trivalent vaccine that we developed, administered with Montanide GEL 01 PR adjuvant, should be conducted on pig farms in the future.
Acknowledgments
We thank Xiaojian Xi for his excellent technical assistance and excellent animal care. This study was supported by the Open Funds of State Key Laboratory of Veterinary Etiological Biology (grant SKLVEB2013KFKT009), the National Natural Science Foundation of China (grants 31302106 and 31001051), and the Research and Development Foundation of Henan University of Science and Technology (grant 2015ZDCXY04).
Footnotes
Conflict of interest statement
None of the authors has any financial or personal relationships that could inappropriately influence or bias the content of the paper.
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