Blood cellular immune response in pigs immunized and challenged with Haemophilus parasuis A.J. Martín de la Fuente a , C.B. Gutiérrez-Martín a, * , J.I. Rodríguez-Barbosa a , S. Martínez-Martínez a , R. Frandoloso a , F. Tejerina b , E.F. Rodríguez-Ferri a a Department of Animal Health, Section of Microbiology and Immunology, Faculty of Veterinary Medicine, University of León, 24007-León, Spain b Department of Veterinary Medicine, Surgery and Anatomy, Section of Reproduction, Faculty of Veterinary Medicine, University of León, 24007-León, Spain article info Article history: Accepted 22 July 2008 Keywords: Haemophilus parasuis Glässer’s disease PBMC Cellular immune response abstract The cellular immune response to an experimental infection by Haemophilus parasuis, the etiological agent of Glässer’s 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 Â 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 + , CD8a + , CD25 + , CD4 + naïve, aIgM + and SWC3 + cells in single-colour fluorescence, and CD4 + /CD8a + and CD8a + /CD8b + 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 (aIgM + ), as well as a significant reduction in CD3 + cells (P < 0.05). These changes were similar for the five groups com- pared, except for the significant increase of CD25 + cells, which was only observed for the bacterin-vacci- nated 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 devel- oped by the different groups was not relevant to protection. Ó 2008 Elsevier Ltd. All rights reserved. 1. Introduction Haemophilus parasuis is a nicotinamide adenine dinucleotide (NAD)-dependent, Gram-negative bacterium of the family Pasteu- rellaceae. It is considered as an early colonizer of the upper respira- tory tract of domestic pigs, but it is better known as the etiological agent of Glässer’s disease. This disease is characterized by a fibrin- ous polyserositis, meningitis and polyarthritis, as well as other conditions, such as septicaemia, myositis or pneumonia (Hoefling, 1991; Amano et al., 1997; Oliveira and Pijoan, 2004). Glässer’s dis- ease has historically been considered a sporadic, stress-associated disease of young pigs; however, since the establishment of specific pathogen free herds, increased spread of the disease and increased mortality rates have been described. In such herds, infection may spread as a contagious disease of high morbidity, being responsible for increased economic losses in the swine industry worldwide (Oliveira and Pijoan, 2004). Initial studies about the immune response developed against H. parasuis have detected antibodies to outer membrane proteins (OMPs) but not against lipopolysaccharide or capsule, thus sug- gesting a more immunogenic role for these proteins (Miniats et al., 1991). Vaccination using bacterins has induced a good pro- tection after challenge with the homologous serotype (Takahaschi et al., 2001); however, a major variability has been reported when testing the development of cross-protection, depending on H. para- suis strains and serotypes (Miniats et al., 1991; Rapp-Gabrielson et al., 1997; Solano-Aguilar et al., 1999; Takahaschi et al., 2001; Bak and Riising, 2002; Hoffman and Bilkei, 2002; Oliveira et al., 2003; Palzer et al., 2007). In addition, no in formation about the po- tential efficacy of modern vaccines based on molecular techniques has been published. With respect to this, recombinant vaccines based on OMPs of Actinobacillus pleuropneumoniae (Rossi-Campos et al., 1992; Goethe et al., 2001) and H. influenzae (Webb and Crip- ps, 1999) have provided a good protection. Similarly, the exposure to a sublethal dose of H. parasuis as immunization procedure has prevented the disease (Oliveira et al., 2001a). On the other hand, little information has been gathered to date on the cellular response developed to H. parasuis, and only leukopenia 0034-5288/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.rvsc.2008.07.010 * Corresponding author. Tel.: +34 987 291 203; fax: +34 987 291 304. E-mail address: cbgutm@unileon.es (C.B. Gutiérrez-Martín). Research in Veterinary Science 86 (2009) 230–234 Contents lists available at ScienceDirect Research in Veterinary Science journal homepage: www.elsevier.com/locate/rvsc