Role of the aspartase in A. pleuropneumoniae infection

In a first experiment, A. pleuropneumoniae ∆aspA was used in an aerosol infection model and compared to the parental strain A. pleuropneumoniae wt. The challenge doses were 3.6 × 10⁴ CFUs (aerosolized for four pigs) in the group challenged with A. pleuropneumoniae wt and 2.2 × 10⁴ CFUs in the group challenged with A. pleuropneumoniae ∆aspA. All animals in this experiment were sacrificed on day 21 post infection. In a second aerosol infection experiment with higher challenge doses, groups of pigs were infected with mutant strains A. pleuropneumoniae ∆aspA or A. pleuropneumoniae ∆aspA∆dmsA (16.38 × 10⁴ CFUs and 11.05 × 10⁴ CFUs for four pigs, respectively) and compared to a control group infected with A. pleuropneumoniae wt (6.76 × 10⁴ CFUs for four pigs). Pigs were sacrificed on day 7, except for three randomly assigned animals in the group challenged with A. pleuropneumoniae ∆aspA∆dmsA and four randomly assigned animals in the control group, which were sacrificed on day 21. Infection with A. pleuropneumoniae wt and both mutants led to an increase in body temperature above 40°C in 38 of the 44 challenged pigs, with no apparent difference observed between the challenge groups. However, dyspnea was observed longer in pigs challenged with A. pleuropneumoniae wt compared to either one of the mutant

strains (Fig. 19A). Animals infected with A. pleuropneumoniae ∆aspA∆dmsA showed a significantly lower clinical score (p < 0.01; Student’s t-Test) in comparison to animals infected with a similar challenge dose of A. pleuropneumoniae ∆aspA or A. pleuropneumoniae wt (Fig. 19B).

Fig. 19: Clinical symptoms in pigs infected with A. pleuropneumoniae wild type and mutant strains. (A) Occurrence of dyspnea in pigs infected with A. pleuropneumoniae wt or mutant strains from days one to seven post infection.

Values are given as percentage of animals showing dyspnea within groups infected with either A. pleuropneumoniae wt (●), A. pleuropneumoniae ∆aspA (■) or A. pleuropneumoniae ∆aspA∆dmsA (▲). (B) Clinical score of pigs infected with different A. pleuropneumoniae strains: A. pleuropneumoniae wt (●), A. pleuro-pneumoniae ∆asp (■), A. pleuropneumoniae ∆aspA∆dmsA (▲). The central symbol in each hourglass shape indicates the geometric mean, the hinges indicate the values in the middle half of the data, and the top and bottom symbols indicate the maximum and minimum values. The asterisk indicates statistical significance (p < 0.01) as determined by the Student's t-Test.

D.2.10.2. Post mortem examination

At necropsy, a lower lung lesion score was seen in the groups challenged with either of the two mutant strains compared to groups challenged with A. pleuro-pneumoniae wt (Table 11) with no apparent differences in the histological examination. Actinobacillus pleuropneumoniae was consistently reisolated from lung lesions in pure culture in 35 of 37 pigs. The number of viable bacteria in pneumonic lung ranged from 10⁵ to 10⁸ CFU/g on day 7 and 10² to 10⁷ CFU/g on day 21 irrespective of the challenge strain. Reisolation of challenge strains from intact lung

succeeded in all animals infected with the high challenge dose of A. pleuro-pneumoniae wt and A. pleuropleuro-pneumoniae ∆aspA, but only in 2 out of 7 pigs infected with a comparable dose of A. pleuropneumoniae ∆aspA∆dmsA (Table 11). Most importantly, using surface swabs, only one and six colonies, respectively, could be isolated from these two animals, in contrast to the several hundred A. pleuro-pneumoniae colonies obtained from a surface smear of intact lung from pigs infected with A. pleuropneumoniae wt or with A. pleuropneumoniae ∆aspA. This finding was supported by the results from quantitative determination of lung colonization. Here, intact lung of pigs infected with either A. pleuropneumoniae wt or A. pleuro-pneumoniae ∆aspA contained up to 10⁶ CFU/g, whereas no bacteria were reisolated from 5 of 7 animals infected with A. pleuropneumoniae ∆aspA∆dmsA; the remaining two animals of this group were found to contain less than 10² CFU/g in intact lung tissue.

Surface smears of palatine tonsils, lymph nodes and heart were sporadically culture positive for A. pleuropneumoniae without consistent differences between the challenge groups.

D.2.10.3. Systemic immune response

Serum samples were obtained one week before and one or three weeks after experimental infection. Two different ELISA systems, using detergent extract or recombinant ApxIIA protein as the solid-phase antigen were employed to quantify the systemic immune response of infected pigs.

All three, A. pleuropneumoniae wt and both mutants induced a strong humoral immune response, and no significant difference was seen in ELISA titers between the different challenge groups on neither day 7 nor day 21 (Table 11).

Table 11: Virulence of A. pleuropneumoniaewt, A. pleuropneumoniae∆aspA and A. pleuropneumoniae∆aspA∆dmsA following aerosol challenge

a Bacterial cultures were grown to the following OD660: 0.37 for A. pleuropneumoniaewt, 0.44 for A. pleuro- pneumoniae∆aspA and 0.45 for A. pleuropneumoniae∆aspA∆dmsA, kept on ice for 10 min and then diluted 300 fold with 150 mM NaCl solution, kept on ice and further diluted 100 fold immediately before challenge; thirteen ml of this suspension were used for challenge in the aerosol chamber. CFUs were determined from challenge dilution. b Day after infection on which animals were sacrificed, post mortem analysis was performed and antibody titers were determined. c The solid phase antigen was prepared as described previously (GOETHE et al. 2000), the number given is the arithmetic mean of the highest serum dilution resulting in an optical density twice as high as the negative control serum at a dilution of 1:100. d Recombinant ApxII was used as solid phase antigen as described previously (LEINER et al. 1999), the number given is the arithmetic mean of the serum activity in ELISA units. e The lung lesion score was determined as described by HANNAN et al. (1982). f Although the lung lesion scores of animals infected with A. pleuropneumoniae∆aspA and A. pleuro- pneumoniae∆aspA∆dmsA are lower than observed for animals infected with A. pleuropneumoniaewt, this difference is not statistically significant with p = 0.11 for A. pleuropneumoniae∆aspA and p = 0.24 for A. pleuro- pneumoniae∆aspA∆dmsA in the Wilcoxon test. g One animal was euthanized on day 6 after infection due to rectal prolapse. This animal was included in lung lesion score and reisolation analyses but excluded from determination of serological response.

D.3. Role of the ferric uptake regulator (Fur) as a global gene