E.F. Rodríguez-Ferri

Blood cellular immune response in pigs immunized and challenged with Haemophilus parasuis

The cellular immune response to an experimental infection by Haemophilus parasuis, the etiological agent of Glässers disease in pigs, was characterized studying changes in peripheral blood mononuclear cells (PBMC) in colostrum-deprived pigs. Five groups were studied, four of those were previously immunized with different formulations and the fifth was maintained as non-immunized control. All groups were challenged with 5 x 10(9) CFU of H. parasuis serotype 5. The non-commercial bacterin conferred a complete protection, while the OMP-vaccine and the exposure to a subletal dose of 10(5) CFU of H. parasuis protected only partially, and the recombinant Tbp B-vaccine induced no protection. PBMC were analyzed using monoclonal antibodies against porcine CD45(+), CD3(+), CD4(+), CD8alpha(+), CD25(+), CD4(+) naïve, alphaIgM(+) and SWC3(+) cells in single-colour fluorescence, and CD4(+)/CD8alpha(+) and CD8alpha(+)/CD8beta(+) combinations in two-colour fluorescence. The different groups showed no significant changes in PBMC subsets following vaccination, and only minor changes were encountered after challenge, consisting mainly of significant increases (P<0.05) in the relative proportions of monocytes and granulocytes (SWC3(+)) and B cells (alphaIgM(+)), as well as a significant reduction in CD3(+) cells (P<0.05). These changes were similar for the five groups compared, except for the significant increase of CD25(+) cells, which was only observed for the bacterin-vaccinated group. These results suggest an increase of trafficking of inflammatory cells and the onset of the adaptive antibody response against H. parasuis infection; in addition, the blood cellular response developed by the different groups was not relevant to protection.

Systemic antibody response in colostrum-deprived pigs experimentally infected with Haemophilus parasuis

The serum antibody response to an experimental infection by Haemophilus parasuis, the etiological agent of Glässers disease in pigs, was characterized by ELISA measuring IgM and IgGt levels against whole-cells and outer-membrane-proteins (OMPs) as antigens. Five groups of pigs were studied, four of those were previously immunized with different formulations, and the fifth was maintained as non-immunized control. All groups were challenged with 5x10(9) CFU of H. parasuis. The non-commercial bacterin induced a full protection against disease, the OMP-vaccine and the exposure to a sublethal dose of 10(5) CFU protected only partially, and the recombinant TbpB-vaccine conferred no protection. The humoral response in the pigs that died after infection (all controls, all those vaccinated with the recombinant TbpB, and two of both those inoculated with OMPs and those exposed to the sublethal dose) could be only measured before it, but it was irrelevant in all cases. However, a specific IgM and IgGt production was observed before challenge in all the surviving pigs, irrespective of the type of immunization received. This antibody response was even greater after H. parasuis infection, especially in those survivors receiving the sublethal dose. These results suggest a role of the antibodies developed after the different immunization protocols in preventing infection and death; therefore, the humoral immunity is protective against experimental Glässers disease.

New insights in cellular immune response in colostrum-deprived pigs after immunization with subunit and commercial vaccines against Glässer’s disease

Four groups of colostrum-deprived pigs were immunized with Porcilis Glässer® (PG) or with subunit vaccines developed by us (rTbpA, NPAPT(M) or NPAPT(Cp)) against Glässers disease, and they were challenged with 3×10(8)CFU of Haemophilus parasuis. A strong reduction in CD3(+)γδTCR(+) cells was seen in non-immunized control and scarcely protected (rTbpA) groups, suggesting that these cells could represent a target of H. parasuis infection. A significant increase in CD172α(+)CD163(+) cells was detected in all groups but PG, while a reduction in SLAIIDR(+) molecules expression was observed after challenge in control animals. Significant increases in CD3ε(+)CD8α(+)CD8β(+) and B cells were detected respectively in control and NPAPT groups, and in scarcely (rTbpA) and well-protected (NPAPT(M) and NPAPT(Cp)) groups. Finally, a greater response in CD4(+)CD8α(-) cells was observed in NPAPT(Cp) compared to NPAPT(M) and PG groups. These results state the potential of NPAPT antigen for developing effective vaccines against Glässers disease.

A vaccine based on a mutant transferrin binding protein B of Haemophilus parasuis induces a strong T-helper 2 response and bacterial clearance after experimental infection.

This study aimed to characterize the type of immune response induced by an experimental vaccine based on a mutant Haemophilus parasuis transferrin binding protein (Tbp) B (Y167A) defective in its ability to bind porcine transferrin. Clinical and pathological signs, bacterial clearance, antibody response and the cytokine profile in alveolar macrophages and spleen after the vaccination and challenge of twenty-two colostrum-deprived pigs with 10(8) CFU of H. parasuis were analysed. Pigs vaccinated with Y167A were compared to those vaccinated with native TbpB (nTbpB), those treated with a commercial bacterin (CB) against Glässers disease, those unvaccinated challenged (CH) and those unvaccinated unchallenged (UNCH) pigs. The rectal temperatures of Y167A pigs resembled those of UNCH pigs and were significantly lower than those of the nTbpB, CB and CH animals. A major reduction in pathological changes of the challenged pigs was observed in the Y167A group. H. parasuis was cleared from 88.9% of the samples from Y167A pigs versus 60.0% and 55.6% from those of the CB and nTbpB groups, respectively. The antibody response elicited by Y167A by ELISA was notably higher than that observed for nTbpB and CB pigs and was capable of preventing the expression and secretion of IL-8. The expression of IL-4 and IL-5, which were associated with the specific antibody levels, suggests that the main mechanism of protection conferred by Y167A vaccine is based on a strong T-helper 2 response.

Comparison of real‐time PCR and culture isolation in colostrum‐deprived pigs immunized and challenged with Haemophilus parasuis

Aims:  A real‐time PCR (RT‐PCR) based on the detection of the infB gene of Haemophilus parasuis is compared with culture isolation (Frandoloso et al., (2011) Clin Vaccine Immunol18, 50–58.), evaluating different subunit or commercial vaccines.

Altered indirect hemagglutination method for easy serotyping of Haemophilus parasuis

Glassers disease is an emergent bacterial disease that affects swine husbandries worldwide causing important economic losses. The aetiological agent, Haemophilus parasuis, is currently divided in fifteen serovars but an increasing number of non-typeable serovars have been reported. Indirect hemagglutination (IHA) is indicated as a serotyping method for H. parasuis. In the present study, we describe an additional step that aims to work around a possible obstacle in the original protocol that may compromise the outcome of this assay. We observed that the choice of anticoagulant for blood collection influences and/or impairs spontaneous adsorption of H. parasuis antigens on sheep red blood cells (SRBCs). However, regardless of the anticoagulant used, chemical treatment of SRBCs with tannic acid induces a stable antigen adsorption (sensitization step). The addition of 1% BSA to SRBCs washing buffer and to antisera dilution augments IHA specificity. Tannic acid treated SRBCs combined with thermo-resistant H. parasuis antigens increases the assay resolution. Thus, our results demonstrate an improvement in the technique of H. parasuis serotyping that will prove valuable to understand Glassers disease epidemiology and to better characterize serovars involved in outbreaks.

Molecular analysis of lungs from pigs immunized with a mutant transferrin binding protein B-based vaccine and challenged with Haemophilus parasuis

The molecular analysis of pigs vaccinated with a mutant transferrin-binding protein B (Y167A) from Haemophilus parasuis was compared with that performed for unvaccinated challenged (UNCH) and unvaccinated unchallenged (UNUN) pigs. Microarray analysis revealed that UNCH group showed the most distinct expression profile for immune response genes, mainly for those genes involved in inflammation or immune cell trafficking. This fact was confirmed by real-time PCR, in which the greatest level of differential expression from this group were CD14, CD163, IL-8 and IL-12. In Y167A group, overexpressed genes included MAP3K8, CD14, IL-12 and CD163. Proteomics revealed that collagen α-1 and peroxiredoxins 2 and 6 were overexpressed in Y167A pigs. Our study reveals new data on genes and proteins involved in H. parasuis infection and several candidates of resistance to infection that are induced by Y167A vaccine. The expression of proinflammatory molecules from Y176A pigs is similar to their expression in UNUN pigs.

Immunogenic characterization of vaccines based on Haemophilus parasuis Nagasaki strain, OmpP2, OmpP5 and OmpD15, in colostrum-deprived pigs experimentally challenged with the same strain

Three recombinant outer membrane proteins (rOmps) from the Haemophilus parasuis Nagasaki strain (serovar 5 reference strain), rOmpP2, rOmpP5 and rOmpD15, which have previously shown protection against H. parasuis infection in mice, were cloned, expressed and evaluated as vaccine antigens in colostrum-deprived pigs. When these animals were immunized with these rOmps and were later challenged intratracheally with 108 CFUs of the Nagasaki strain, no protection was seen in terms of survival, clinical signs, pathological results and recovery of H. parasuis. We hypothesized that a possible explanation for this lack of protection could be the low number of epitopes accessible to the immune system as a consequence of their poor exposure on the bacterial surface so that the immune response would not be able to protect against experimental infection by H. parasuis when a fully susceptible animal model, such as pigs, was used.

Molecular study of an outer fragment of Haemophilus parasuis neuraminidase and utility with diagnostic and immunogen purposes

Haemophilus parasuis is a swine pathogenic organism, being the causative agent of Glässers disease. It has got some virulence factors, some of which act as potential candidates for the vaccine developing. Among them there is the neuraminidase enzyme, which is located inside the outer membrane and contains a β-barrel domain with seven external loops. By using the polymerase chain reaction technique, the β-barrel fragment was amplified, sequenced and analysed for the 15 H. parasuis reference serotypes. The results showed a small diversity for them, except for serotype 2, which has a deletion that covers the loops with potential to be used as vaccine antigen. However, some of the other serotypes showed the same nucleotidic sequence between them, such those 6 and 7 or those 12 and 13. This fact was also confirmed by means of phylogenetic analysis. For these reasons, the tested fragment might result in a putative candidate for the development of subunit vaccines against all the serotypes causing Glässers disease outbreaks, with the exception of serotype 2, alone or in combination with other proven immunogenicity molecules. Anyway, further studies should be carried out in pigs in order to confirm this hypothesis. Finally, this outer fragment of H. parasuis neuraminidase could be used as a suitable diagnostic tool at a species level, for instance, by PCR.