Acute and subacute response of iron, zinc, copper and selenium in pigs experimentally infected with Actinobacillus pleuropneumoniae.

This study was performed to characterise the response of iron (Fe), zinc (Zn), copper (Cu) and selenium (Se) in bacterial-induced porcine acute phase reaction (APR). Twenty piglets were challenged by aerosolic infection with Actinobacillus pleuropneumoniae (A.pp.) serotype 2, ten piglets serving as controls. Blood sampling was done initially and at day 4 and 21 after infection, collection of liver tissue was done at day 21 (autopsy). A.pp.-infection caused fever and respiratory symptoms. APR at day 4 after infection was marked by an increase in total white blood cells, granulocytes and monocytes in whole blood samples and an increase in globulin/albumin ratio (G/A), α2-globulins, C-reactive protein, haptoglobin, ceruloplasmin (Cp), Cu and Se in serum. Concurrently, there was a decrease in haemoglobin (Hb) and packed cell volume (PCV) in whole blood as well as a decrease in albumin, transferrin, total iron binding capacity and Fe in serum and Zn in plasma. The subacute stage at day 21 was characterised by progressively increased concentrations of G/A, ß-globulins and γ-globulins reflecting the specific immune reaction. Hb and PCV showed further decreases, all other parameters returned to the initial concentrations. Glutathione peroxidase activity in plasma and liver tissue remained unaffected by A.pp.-infection. The liver concentration (day 21) of Zn was found to be higher, that of Se was lower in the A.pp.-group, whereas hepatic concentrations of Cu and Fe were not affected by A.pp.-infection. In summary, the acute and subacute stages of A.pp.-infection were accurately characterised by the APR-related parameters. Se was only marginally affected by the A.pp.-infection. The elevated plasma Cu concentration may be a side effect of the transient hepatic induction of Cp synthesis. Zn responded, being distinctly reduced in plasma and probably having been sequestered in the liver tissue. Reduction in serum Fe can be regarded as an unspecific defence mechanism in A.pp.-infection to withdraw Fe from bacterial acquisition systems.

Analysis of the Actinobacillus pleuropneumoniae HlyX (FNR) regulon and identification of iron-regulated protein B as an essential virulence factor.

The Gram-negative rod Actinobacillus pleuropneumoniae is a facultative anaerobic pathogen of the porcine respiratory tract, and HlyX, the A. pleuropneumoniae homologue of fumarate and nitrate reduction regulator (FNR), has been shown to be important for persistence. An A. pleuropneumoniae hlyX deletion mutant has a decreased generation time but highly prolonged survival in comparison to its wild type parent strain when grown anaerobically in glucose-supplemented medium. Applying a combination of proteomic and transcriptomic approaches as well as in silico analyses, we identified 23 different proteins and 418 genes to be modulated by HlyX (> or = twofold up- or down-regulated). A putative HlyX-box was identified upstream of 54 of these genes implying direct control by HlyX. Consistent with its role as a strong positive regulator, HlyX induced the expression of genes for anaerobic metabolism encoding alternative terminal reductases and hydrogenases. In addition, expression of virulence-associated genes encoding iron uptake systems, a putative DNA adenine modification system, and an autotransporter serine protease were induced by HlyX under anaerobic growth conditions. With respect to virulence-associated genes, we focused on the iron-regulated protein B (FrpB) as it is the outer membrane protein most strongly up-regulated by HlyX. An frpB deletion mutant of A. pleuropneumoniae had the same growth characteristics as wild type grown aerobically and anaerobically. In contrast, A. pleuropneumoniae DeltafrpB did not cause any disease and could not be re-isolated from experimentally infected pigs, thereby identifying FrpB as a previously unknown virulence factor.