Expression levels of immune markers in Actinobacillus pleuropneumoniae infected pigs and their relation to breed and clinical symptoms

Abstract

Background: In pigs little is known about the role of innate immune defence in bacterial infections of the respiratory tract, despite their major role in pig production. In the present study we characterized and compared in vitro and in vivo activation of immune markers of different pig breeds 7 days before, and 4 and 21 days after an experimental aerosol infection with Actinobacillus (A.) pleuropneumoniae.

Results: In vitro stimulation of bronchoalveolar lavage fluid (BALF) and blood leukocytes with A. pleuropneumoniae, Streptococcus suis, PMA and LPS led to production of different amounts of H2O2, NO and TNF-alpha, depending on the stimulus, individual, breed and time of infection. Generally, significant responses to in vitro stimulation were observed only in blood leukocytes, whereas the alveolar macrophages showed a high basal activation. In addition, the production of haptoglobin and cytokines (TNF-alpha, IFN-gamma and IL-10) in vivo was measured in plasma and BALF. Plasma haptoglobin levels mirrored the clinical manifestations at 4 days post-infection. In plasma and BALF TNF-alpha could not be detected, whereas variable levels of IFN-gamma were found at pre- and post-infection times. IL-10 was found in some plasma but in none of the BALF samples. The different expression levels in individuals within the breeds correlated for some markers with the severity of clinical manifestations, e.g. H2O2, plasma haptoglobin and BALF IFN-gamma for German Landrace pigs.

Conclusion: Our findings revealed differences in the activation of the immune markers with respect to infection time, individuals and breeds. Moreover, results showed different correlation grades between the immune markers produced in vitro or in vivo and the clinical manifestations. Further analyses will have to show whether these markers may serve as correlates of protection against porcine respiratory infections.

Figure 1

Kinetics of the activation of immune markers in vitro in response to A. pleuropneumoniae infection. H₂O₂ (A) and TNF-α (B) production were measured after in vitro stimulation of blood leukocytes with PMA (for A) or inactivated A. pleuropneumoniae (for B). (C) Basal expression of TNF-α by BALF leukocytes after 6 h cultivation in vitro. The expression of the specific markers was recorded as shown on the bottom of the graph for each breed at 7 days before infection (-7) as well as at 4 and 21 days after infection. The box represents the 50% between 25% and 75% quartiles. The line inside the box indicates the median. The top and bottom lines denote the 5 and 95 percentile, whereas the black crosses denote minimum and maximum values. The numbers on the top of the boxes indicate the number of samples examined. ND – not determined; * p < 0.05; ** p < 0.01; *** p < 0.001.

Figure 2

Kinetics of the activation of immune markers in vivo in response to A. pleuropneumoniae infection. The concentrations of haptoglobin in plasma (A) and of IFN-γ in plasma (B) and BALF (C) were measured at 7 days before infection (-7) as well as at 4 and 21 days after infection. The box represents the 50% between 25% and 75% quartiles. The line inside the box indicates the median. The top and bottom lines denote the 5 and 95 percentile, whereas the black crosses denote minimum and maximum values. The numbers on the top of the boxes indicate the number of samples examined. ND – not determined; * p < 0.05; ** p < 0.01; *** p < 0.001.

Figure 3

Linear regression analysis of the clinical scores of the German Landrace, Pietrain, Hampshire and Large White pigs versus plasma concentration of haptoglobin at 4 days after infection. Each point on the graph represents an individual pig value.