Immunogenicity of an Autogenous Streptococcus suis Bacterin in Preparturient Sows and Their Piglets in Relation to Protection after Weaning

Pig herd.All piglets investigated in this study were from a single closed farrow-to-finish farm with 105 sows with a history of S. suis problems in weaning piglets and growers. Immunization of pigs against S. suis started with this study and was performed only with the S. suis bacterin described below. All sows received porcine circovirus 2 vaccination (Circovac; Merial, Germany) 5 weeks antepartum and Escherichia coli and Clostridium perfringens type C immunization (Enterisol Coli-Clost; Boehringer Ingelheim, Germany) 3 weeks antepartum. Seven days postpartum, sows were immunized against parvovirus and Erysipelothrix rhusiopathiae (Parvoruvac, Merial, Germany). Fourteen days postpartum, sows and suckling piglets were vaccinated against porcine respiratory and reproductive syndrome virus (Ingelvac PRRS MLV; Boehringer Ingelheim, Germany). Furthermore, a Mycoplasma hyopneumoniae vaccine (Stellamune Mykoplasma; Pfizer, Germany) was applied to suckling piglets at ages 5 and 26 days.

Weaning was performed in the 4th week postpartum. Cross-fostering was not practiced with the piglets included in this study. Five days before challenge, respective piglets were transported to the institute for experimental infection under safety level 2 laboratory conditions.

Bacterial strains and growth conditions. S. suis strain Br3/6 is an mrp⁺epf* sly⁺cps2 strain which was isolated from the brain of a piglet of this particular herd with severe fibrinosuppurative meningitis. Strain 10 is an mrp⁺epf⁺sly⁺cps2 reference strain which has been shown to be highly virulent in experimental infections of piglets (2, 21). A3286/94 is an mrp* sly+ serotype 9 S. suis strain of sequence type 99 which was originally isolated from a pig with meningitis (6).

Preparation of the bacterin.The bacterin was generated with S. suis strain Br3/6 grown overnight in Bacto Todd-Hewitt broth (Becton Dickinson Diagnostics, Heidelberg, Germany) at 37°C and inactivated in 0.1% formaldehyde (no centrifugation of the culture). Emulsigen was added as an adjuvant (20% [vol/vol]). Each immunization dose contained approximately 10⁹ CFU. The mixtures were stirred for 8 h, screened for sterility, and stored at 8°C.

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FIG. 1.

Experimental design of this study for comparative evaluation of autogenous S. suis serotype 2 bacterin vaccination regimes.

In the first two experiments, sows in the same gestation stage (5 weeks antepartum) were randomly divided into an S. suis immunization group and a control group. The two groups were housed in the same unit. The only difference in treatment between the two groups was application of the S. suis bacterin in the 5th and 3rd weeks antepartum. After the 2nd experiment, all sows of this particular herd were immunized with the S. suis bacterin in the 5th and 3rd weeks antepartum. The sows (and piglets) investigated in this herd had not been vaccinated against S. suis before inclusion of these animals in this study. Piglets were vaccinated either in the 2nd and 4th weeks postpartum (1st and 2nd experiments) or in the 4th and 6th weeks postpartum (3rd experiment).

In the 1st experiment, all piglets used for immunization and subsequent challenge were from different sows (either immunized or nonimmunized). In the 2nd and 3rd experiments, all piglets of a sow were divided randomly into two groups, one used for S. suis immunization and the other as a control. The two groups were not separated physically. For challenge, two piglets, one vaccinated and one nonvaccinated, from each sow were used (randomly selected among healthy and well-developed piglets).

Challenge experiments.Sixty piglets from a single herd described above were infected experimentally and cared for in accordance with the principles outlined in the European Convention for the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes (European Treaty Series, no. 123: http://conventions.coe.int/treaty/EN/Treaties/html/123.htm ANX; permit no. 33.9-42502-04-07/1395 and 09/1684). The piglets were challenged intravenously with 1 × 10⁸ CFU of strain Br3/6 either at an age of 6 weeks (1st experiment) or at an age of 8 weeks (2nd and 3rd experiments). The health status of the animals was monitored every 6 to 10 h after challenge. In the case of high fever (≥40.5°C), apathy as well as anorexia, and in all cases of clinical signs of acute polyarthritis or severe meningitis, animals were euthanized for reasons of animal welfare. All surviving piglets were sacrificed 9 days postinfection.

Serum and colostrum samples.Serum samples were collected from all sows 5 weeks and 1 week antepartum (pre- and postvaccination, respectively). Colostrum was obtained within 4 and 6 h after the start of birth and frozen immediately at −20°C.

In the 1st experiment, serum was collected from piglets in the 2nd, 4th, and 6th weeks postpartum (pre-1st, pre-2nd, and postvaccination, respectively). In the 2nd and 3rd experiments, an additional serum sample was collected in the 8th week postpartum (all samples prior to experimental infection).

Histopathological and bacteriological screening.The histological screenings were carried out and scored with blinded experiments as described previously (2). All tissues screened histologically were also investigated bacteriologically through culture- and PCR-based detection of the challenge strain (20).

Detection of MRP-specific antibodies.MRP-specific IgG (IgG1 and IgG2) was determined by ELISA as described previously (3, 13). Based on the information of the manufacturer of the mouse anti-porcine IgG1 and IgG2 antisera (Serotec; Kidlington, Oxford, United Kingdom), the definition of subclasses was based upon electrophoretical mobility, with IgG1 migrating faster than IgG2 as described by Metzger and Fougereau (16). However, electrophoresis does not allow differentiation of the six porcine IgG subclasses described more recently on the basis of genomics (7, 12). Since there are currently no purified standards for the different IgG variants, it is impossible to determine the subclasses or allotypic variants of subclasses the two antisera recognize (J. E. Butler, personal communication).

Briefly, the anti-MRP ELISA was performed with Maxisorb plates (Nunc, Rochester, NY) coated with 5 μg recombinant MRP (rMRP) or bovine serum albumin (BSA) (background measurement) in carbonate buffer. Mouse anti-porcine IgG1 and IgG2 antisera (Serotec) and peroxidase-conjugated goat anti-mouse IgG antiserum (Dianova, Hamburg, Germany) were used as primary and secondary antibodies, respectively. The plates were developed with 2,2-azino-di-[3-ethylbenzithiazoline sulfonate] (ABTS) (Boehringer, Mannheim, Germany) and 0.002% H₂O₂ as a substrate. Absorbance was measured at 405 nm. Every serum sample and the controls were measured in a duplicate series of 4 (reference serum, 7) 2-fold dilutions in phosphate-buffered saline-Tween 20 (PBST) (starting with 1:200). A convalescent-phase serum obtained from a piglet 20 days after experimental infection with the MRP⁺ EF⁺ serotype 2 reference strain 10 was used as a standard and defined to have 100 ELISA units. Optical densities were converted to antibody concentrations through log linear regression analysis after background subtraction. The ELISA units of each sample were defined as the mean of the calculated units for each of the four dilutions of the two series. Data were considered only if they met the following criteria: deviation of duplicates of <15%, slope of the linear portion of the reference standard curve between 0.8 and 1.2 for IgG1 and between 1 and 1.4 for IgG2, correlation coefficient between 0.9 and 1.0, and controls within established ranges.

Purification of serotype 2 capsular polysaccharides (CPS2) and detection of CPS2-specific antibodies.Purification of CPS2 and detection of CPS2-specific IgG antibodies in an ELISA were performed as described previously (3). In this ELISA, a peroxidase-conjugated goat anti-swine IgG (heavy plus light chains) antiserum (Dianova, Hamburg, Germany) was used.

Opsonophagocytosis assay.Opsonophagocytic killing in the presence of 20% (vol/vol) porcine serum was assayed as described previously (3). The same batch of frozen glycerol stocks of an S. suis strain Br3/6 culture (logarithmic growth phase) was used during the whole experiment (13, 19). S. suis strain Br3/6 was incubated with porcine neutrophils at a starting multiplicity of infection of 0.35 (1.75 × 10⁶ CFU incubated with 5 × 10⁶ neutrophils). The survival factor represented the ratio of CFU at 1 h to CFU at time zero. Results of an opsonophagocytosis assay were accepted only if survival factors of strain Br3/6 were below 0.2 in the positive control (porcine hyperimmune serum against BSA-conjugated serotype 2 capsular polysaccharides) and above 1.5 in the negative control (serum from gnotobiotic piglets). The serotype 9 strain A3286/94 was included as a control to confirm specificity of killing mediated by the positive-control serum (survival factor above 1.2).

Statistical analysis.Statistical analysis with the Mann-Whitney test was performed to analyze differences between the different groups of piglets used in each experiment. The Wilcoxon test was used for comparison of different time point values within the same group. The data in the Kaplan-Meyer survival diagrams were analyzed with the log rank test. Probabilities lower than 0.05 were considered significant.

Characterization of S. suis bacterin immunogenicities in sows.A main objective of this work was to evaluate immunogenicity of a bacterin applied 5 and 3 weeks antepartum in dams, since this had not been investigated previously. In all three experiments, vaccination of sows with the autogenous S. suis bacterin elicited a significant IgG serum response against the surface-associated protein MRP, which was used as an antigen in respective ELISAs (IgG1 and IgG2; see Materials and Methods). Serum IgG anti-MRP titers were significantly higher in vaccinated sows than in nonvaccinated sows of the first two experiments (Fig. 2 A; see also Fig. S1A in the supplemental material). Accordingly, colostrum from immunized sows contained significantly higher titers of anti-MRP IgG than colostrum from nonvaccinated animals (Fig. 2B; see also Fig. S1B). The MRP-specific IgG1 and IgG2 titers were about twice as high in colostrum than in sera 1 week antepartum (e.g., for vaccinated sows of the 1st experiment: median ELISA units for IgG1 and IgG2 of 839 and 2098 for colostrum and 508 and 946 for serum, respectively; Fig. 2A and B).

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FIG. 2.

Detection of MRP- and CPS2-specific antibodies in sows of the 1st experiment vaccinated 5 and 3 weeks antepartum with a bacterin of S. suis Br3/6 (mrp⁺epf* sly⁺cps2). Serum samples were collected 5 weeks (pre) and 1 week (post) antepartum. (A) Serum anti-MRP IgG1 and IgG2 responses (see Materials and Methods). (B) Comparison of MRP-specific titers in colostrum of vaccinated (vac) and nonvaccinated (non-vac) sows. (C) Serum total IgG responses against purified serotype 2 capsular polysaccharides.

Application of bacterin to weaning piglets did not elicit a prominent immune response against serotype 2 capsular polysaccharides in our previous study (3). Since immunogenicity of capsular polysaccharides might be greater in older animals, the sera of the sows of the 1st experiment were also screened with respect to CPS2-specific antibodies. As shown in Fig. 2C, bacterin application elicited a significant total IgG response against CPS2 in serum from preparturient sows (P = 0.028). This resulted in significantly higher anti-CPS2 titers for vaccinated sows 1 week antepartum than for unvaccinated sows (P = 0.049).

In our previous study, we demonstrated correlation of induction of opsonizing antibodies with protection in S. suis immunization (3). Therefore, titers of opsonizing antibodies were also determined in this study. As shown in Fig. 3, immunization of sows induced opsonizing antibodies antepartum, leading to sufficient killing of the challenge strain by porcine neutrophils in the presence of post- but not prevaccination serum (P = 0.0001). In accordance, titers of opsonizing antibodies in sera from vaccinated sows against the homologous serotype 2 S. suis strain were significantly higher than respective titers for unvaccinated sows (1 week antepartum; P = 0.00004) (Fig. 3).

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FIG. 3.

Opsonophagocytic killing by porcine neutrophils in the presence of serum of sows (data from all three experiments). The y axis depicts the survival factor of the S. suis strain in the opsonophagocytosis assay with porcine neutrophils and sow serum, which is reciprocal to the titer of opsonizing antibody. Bacterin application was performed 5 and 3 weeks antepartum.

In conclusion, quantitative (anti-MRP and anti-CPS2 titers) and functional (opsonization) analysis of humoral immunity against S. suis serotype 2 in sows indicated induction of a strong and efficient immune response after bacterin vaccination 5 and 3 weeks antepartum. Analysis of humoral immunity in suckling and weaning piglets and challenge experiments were performed to investigate if the induced maternal immunity interfered with active immunization of suckling (1st and 2nd experiments) and weaning (3rd experiment) piglets and if the maternal immunity provided protection in 6-week-old (1st experiment) and 8-week-old (2nd experiment) piglets.

Protective efficacies of sow and suckling piglet vaccination in weaning piglets.In the 1st experiment, vaccination of suckling piglets with an autogenous S. suis bacterin was performed alone or in combination with sow immunization (Fig. 1). Application of the bacterin to suckling piglets only did not result in significant protection against morbidity after S. suis challenge 2 weeks after weaning (P = 0.2) (Fig. 4 A). In contrast, weaning piglets from vaccinated sows, which were immunized as suckling piglets, showed less morbidity after homologous challenge than respective control piglets (P = 0.009) (Fig. 4A and Table 1). Accordingly, pathohistological lesions were also less severe in these piglets (Table 2). Furthermore, the challenge strain was detected in only 1 tissue sample from each of 3 piglets weaned from vaccinated sows, in contrast to detection of the challenge strain in a total of 11 tissue samples collected from 5 different control piglets (Table 3).

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FIG. 4.

Kaplan-Meyer morbidity diagrams of piglets challenged intravenously with 10⁸ CFU of S. suis Br3/6 (mrp⁺epf* sly⁺cps2) after application of different vaccination regimes, including sow (5 and 3 weeks antepartum) as well as suckling and weaning piglet vaccination with an autogenous bacterin. (A) Challenge in the 6th week postpartum after suckling piglet vaccination (2nd and 4th weeks postpartum) combined in one group with sow immunization (1st experiment). (B) Challenge in the 8th week postpartum after different vaccination regimes, including sow and suckling piglet immunization (2nd and 4th weeks postpartum, 2nd experiment). (C) Challenge in the 8th week postpartum after vaccination of sows combined in one group with weaning piglet vaccination (4th and 6th weeks postpartum, 3rd experiment).

MRP-specific IgG titers were significantly higher for piglets from vaccinated sows in the 2nd, 4th, and 6th weeks postpartum than for piglets of unvaccinated sows (IgG1 and IgG2; Fig. 5 A). A significant increase in mean MRP-specific IgG titers was not observed in any of the three groups of the 1st experiment (Fig. 1 shows the experimental design, and Fig. 5A shows results). Twelve days postvaccination, there was a significant difference in titers of MRP-specific IgG1 but not IgG2 between the group of vaccinated piglets raised by unvaccinated sows and the control group (P = 0.042) (Fig. 5A). However, 5 of these 6 vaccinated piglets had IgG1 titers below 40 ELISA units, which were based on controls of the ELISA not considered to be clearly positive.

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FIG. 5.

MRP-specific IgG1 and IgG2 (A) or opsonizing antibody (B) titers in serum of piglets with or without sow vaccination and their course after suckling piglet immunization in the 2nd and 4th weeks postpartum (data of the 1st experiment). The y axis in panel B depicts the survival factor of the S. suis strain in the opsonophagocytosis assay with porcine neutrophils and piglet serum, which is reciprocal to the titer of opsonizing antibody.

Titers of opsonizing antibodies determined in neutrophil killing assays were significantly higher in the 2nd week postpartum for piglets from vaccinated sows than for piglets from nonvaccinated sows (Fig. 5B) but not comparable to the high titers detected in the sera of the vaccinated sows (Fig. 3). At 6 weeks postpartum, titers of opsonizing antibodies had declined significantly in suckling piglets from vaccinated sows, resulting in comparable titers of opsonizing antibodies for all three groups.

In conclusion, autogenous bacterin vaccination of suckling piglets failed to elicit a significant MRP-specific serum IgG response or an increase in titers of opsonizing antibodies. A significant protection of respective 6-week-old piglets against morbidity after homologous experimental challenge was observed only in piglets from sows vaccinated 5 and 3 weeks antepartum. The most likely explanation for this protection was protective passive maternal immunity in these animals. To test this, experiment 2 was conducted with challenge in older piglets.

Protective efficacies of sow and suckling piglet vaccination in growers.The objective of the 2nd experiment was to evaluate different bacterin vaccination regimes with sows and suckling piglets for protection in growers. As shown in Table 1 and Fig. 4B, morbidity and mortality of 8-week-old piglets were very high and very much alike in all groups. In agreement, the mean pathohistological score of all groups was above 2.5, in contrast to results in the 1st experiment, which included challenge of 6- instead of 8-week-old piglets (Table 2). In addition, all groups of the 2nd experiment included piglets, which allowed detection of the challenge strain in multiple samples, such as cerebrospinal and joint fluids (Table 3).

A significant decline of MRP-specific IgG1 and IgG2 titers was observed from the 2nd to 4th and 4th to 8th weeks postpartum for piglets from vaccinated sows (P = 0.028 or 0.043) (see Fig. S2 in the supplemental material). This decline was observed irrespective of bacterin application during the suckling period (no significant differences). The theoretical half lives of MRP-specific IgG1 and IgG2 calculated from the declines observed from the 2nd to the 4th week postpartum for the unvaccinated suckling piglets born from vaccinated sows were 6.9 days (standard deviation [SD] = 1.1) and 6.7 days (SD = 1.2), respectively. In the group of piglets derived from nonvaccinated sows with bacterin application in the 2nd and 4th weeks postpartum, a slight but significant increase of MRP-specific IgG2 but not IgG1 was observed from the 4th to 8th wee postpartum (P = 0.046) (see Fig. S2). At an age of 8 weeks, the mean MRP-specific IgG1 and IgG2 titers for these piglets, which were vaccinated during the suckling period, were 45 and 92 ELISA units and thus only slightly higher than those for the unvaccinated control group, with mean values of 16 and 52 ELISA units, respectively (P = 0.016 and 0.1, respectively). None of the vaccination regimes tested in the 2nd experiment induced opsonizing antibodies in piglets at ages of 6 and also 8 weeks (mean survival factors of S. suis strain Br3/6 were between 1.3 and 1.5 in the opsonophagocytosis assay with no significant differences).

In conclusion, neither application of S. suis bacterin to preparturient sows nor that to suckling piglets or both elicited protection in 8-week-old piglets. The detected positive MRP-specific IgG2 titers were not associated with opsonizing activity. The lack of opsonizing antibodies most likely explains the lack of protection.

Protective efficacies of sow and weaning piglet vaccination in growers.The objective of the 3rd experiment was to assess if active immunization of piglets weaned from bacterin-vaccinated sows can elicit an active immune response and protection (using the same S. suis bacterin). In contrast to the previous experiments, the bacterin was applied in the 4th and 6th weeks instead of the 2nd and 4th weeks postpartum (Fig. 1). As demonstrated in Fig. S3A in the supplemental material, MRP-specific IgG1 and IgG2 titers declined from the 2nd to the 4th and the 4th to the 8th week postpartum for nonvaccinated piglets and for vaccinated piglets (with the exception of one vaccinated piglet). However, MRP-specific IgG1 but not IgG2 titers were significantly higher for vaccinated piglets than for nonvaccinated piglets at an age of 8 weeks (P = 0.015 and 0.83, respectively). Application of the bacterin to weaning piglets did not elicit significant titers of opsonizing antibodies against the homologous serotype 2 strain (P = 0.51 for comparison of pre- and post-vaccination sera and P = 0.085 for comparison of sera from vaccinated and nonvaccinated 8-week-old piglets) (see Fig. S3B). In agreement, morbidity and mortality were above 50% in vaccinated and nonvaccinated piglets challenged with this strain, with no significant differences between the two groups (Table 1 and Fig. 4C). Differences between the two groups also were not recorded with respect to pathohistological alterations and detection of the challenge strain in tissue samples (Tables 2 and 3). In conclusion, quantitative and qualitative serological investigations of bacterin-vaccinated piglets weaned from bacterin-vaccinated sows indicated a lack of a prominent active immune response in these weaned piglets, in agreement with the lack of protection observed in the challenge experiment.