A crude capsular polysaccharide extract as a potential novel subunit vaccine with cross-protection against the most prevalent serovars of Glaesserella (Haemophilus) parasuis in the Czech Republic

Abstract

Glaesserella (Haemophilus) parasuis is a part of the normal flora of the respiratory tract of pigs. However, under certain conditions it can also induce severe systemic disease with high morbidity and mortality leading to gross economic losses in the pig industry. The most prevalent serovars in pig herds in the Czech Republic are the virulent serovars 1, 4, 5 and 13. The currently available commercial vaccines are inactivated vaccines with certain limitations, such as no or poor cross-serovar protection. Therefore, the aim of the present study was to construct a subunit vaccine with a crude capsular polysaccharide extract (cCPS) isolated from G. parasuis CAPM 6475 (serovar 5) and evaluate its immunogenicity in a mouse model. Mice were immunised subcutaneously with two doses of the constructed vaccine in a 14-day interval and challenged intraperitoneally with various G. parasuis strains (serovars 1, 4, 5, 13) at 21 days after the second immunisation. The results of the ELISA test showed that the boost dose of the vaccine induced the production of IgG antibodies in high levels. On the basis of the death cases, the pathological findings and the bacterial isolation, the mice immunised with the cCPS were partially protected against the challenge with the homologous serovar 5 as well as with heterologous serovars 1, 4 and 13 of G. parasuis. The cross-reaction of the mixed serum from the immunised mice with the tested serovars was seen in the western-blotting also. Moreover, the most abundant protein found in the cCPS by mass spectrometry was catalase, a protein of molecular weight 55 kDa that may correspond to the strongest reaction seen in the western-blotting. Our findings indicated that the crude capsular polysaccharide extract may provide an effective immunogenicity in preventing a G. parasuis infection caused by the most prevalent serovars in the Czech Republic. However, the evaluation of the efficacy needs to be performed in pigs before any conclusions can be drawn.

The Role of Antibodies Against the Crude Capsular Extract in the Immune Response of Porcine Alveolar Macrophages to In Vitro Infection of Various Serovars of Glaesserella (Haemophilus) parasuis

“…To determine whether IgG antibodies against the crude capsular extract of the virulent G. parasuis strain CAPM4675 increase phagocytosis of various G. parasuis strains in vitro , we isolated IgG from sera of the cCE-immunized pigs, incubated different G. parasuis strains with these IgG and subsequently infected PAMs. We used these antibodies because mice immunized with the cCE in our previous study were partially protected against the challenge with various G. parasuis strains and the anti-cCE antibodies in mice sera were able to bind to various strains in the Western-blotting ( 13 ). In addition, we proved the specific reactivity of the post-vaccinal antibodies from positive pig sera with the capsular extract as well as with various G. parasuis strains also by the Western-blotting, TEM and immunoprecipitation method.…”

“…The preparation method of the crude capsular extract (cCE) obtained from G . parasuis strain CAPM 6475 by methanol extraction was described in our previous study ( 13 ). Briefly, the overnight culture of G. parasuis resuspended in D-PBS was incubated in a water bath at 60°C for 1 h and formaldehyde was added to the bacterial suspension to a final concentration of 0.2% (v/v).…”

“…The antibody titer against the cCE in the negative or the positive sera of pigs was determined by in house indirect enzyme-linked immunosorbent assay (ELISA) ( 13 ). The cCE antigen was dissolved in a carbonate-bicarbonate buffer (pH 9.6) in a concentration of 1 μg/mL that was subsequently dispensed (100 μL/well) into the wells of the Nunc MaxiSorp ® flat-bottom 96-well polystyrene microtiter plate (ThermoFisher Scientific).…”

“…In our previous study, the cCE was highly immunogenic and mice immunized with the cCE were partially protected against the challenge with various G. parasuis strains. Moreover, mice immunized with the cCE decreased bacterial load in the target tissues comparing to the non-immunized mice ( 13 ). The objective of the present study was to determine the role of antibodies against this capsular extract in the immune response of PAMs to in vitro infection with various G. parasuis strains opsonized by these antibodies.…”

“…Since it has been known that G. parasuis is resistant to serum complement (11) and bacterial capsule interferes with complement deposition (12), opsonization of the bacterium would be more effective by antibodies (7). Based on these facts, we decided to prepare the crude capsular extract (cCE) from the virulent G. parasuis strain that contains capsular polysaccharides and also proteins associated with the virulence of the bacterium (13) and thus antibodies against this antigens may opsonize the bacterium more efficiently. The protective role of antibodies against G. parasuis infection was proved in numerous in vivo experiments (14)(15)(16).…”