Use of an Actinobacillus pleuropneumoniae multiple mutant as a vaccine that allows differentiation of vaccinated and infected animals

Abstract

Vaccination against Actinobacillus pleuropneumoniae is hampered by the lack of vaccines inducing reliable cross-serotype protection. In contrast, pigs surviving natural infection are at least partially protected from clinical symptoms upon reinfection with any serotype. Thus, we set out to construct an attenuated A. pleuropneumoniae live vaccine allowing the differentiation of vaccinated from infected animals (the DIVA concept) by successively deleting virulence-associated genes. Based on an A. pleuropneumoniae serotype 2 prototype live negative marker vaccine (W. Tonpitak, N. Baltes, I. Hennig-Pauka, and G.-F. Gerlach, Infect. Immun. 70:7120-7125, 2002), genes encoding three enzymes involved in anaerobic respiration and the ferric uptake regulator Fur were deleted, resulting in a highly attenuated sixfold mutant; this mutant was still able to colonize the lower respiratory tract and induced a detectable immune response. Upon a single aerosol application, this mutant provided significant protection from clinical symptoms upon heterologous infection with an antigenically distinct A. pleuropneumoniae serotype 9 challenge strain and allowed the serological discrimination between infected and vaccinated groups.

FIG. 1.

PCR analyses of A. pleuropneumoniae wild-type and isogenic mutant strains. Lanes: 1, A. pleuropneumoniae serotype 2 wild type; 2, A. pleuropneumoniae ΔapxIIAΔureCΔdmsA; 3, A. pleuropneumoniae ΔapxIIAΔureCΔdmsAΔhybB; 4, A. pleuropneumoniae ΔapxIIAΔureCΔdmsAΔhybBΔaspA; 5, A. pleuropneumoniae ΔapxIIAΔureCΔdmsAΔhybBΔaspAΔfur. Lanes M, size marker. The numbers to the left indicate the size of the PCR products obtained. Primers used were oDMSAdel1 and oDMSAdel2 (for the dmsA gene), o34-1f and 034-1r (for the hybB gene), oASPX and oASPY (for the aspA gene), and oFURX and oFURY (for the fur gene).

FIG. 2.

Lung lesion score upon challenge with different mutant strains of A. pleuropneumoniae serotype 2. The central symbol within the hourglass shape represents the geometric mean, the hinges present the values in the middle of each half of data, and the top and bottom symbols mark the minimum and maximum value. The asterisk denotes statistical significance (P < 0.05) in the Wilcoxon signed-rank test.

FIG. 3.

Antibody titer upon heterologous challenge. Shown are humoral immune responses of control and vaccinated pigs on the day before and 21 days after infection, assessed using a detergent extract (deELISA), recombinant TbpB protein of A. pleuropneumoniae serotype 7 (TbpB-ELISA), recombinant ApxIV protein (ApxIV-ELISA), and the recombinant ApxIIA protein (ApxII-ELISA) as solid-phase antigen. The immune response was expressed in ELISA units (based on an external standard) for the standardized ApxII-ELISA, with activities of ≥25 ELISA units considered positive. Using the standardized ApxIV-ELISA, activities of ≥40% in comparison to an external control were considered positive; for the deELISA and the TbpB-ELISA, the immune response was expressed as a serum titer in comparison to that of an internal negative control.