Pathway deregulation and expression QTLs in response to Actinobacillus pleuropneumoniae infection in swine.

Actinobacillus (A.) pleuropneumoniae is among the most important pathogens in pig. The agent causes severe economic losses due to decreased performance, the occurrence of acute or chronic pleuropneumonia, and an increase in death incidence. Since therapeutics cannot be used in a sustainable manner, and vaccination is not always available, new prophylactic measures are urgently needed. Recent research has provided evidence for a genetic predisposition in susceptibility to A. pleuropneumoniae in a Hampshire × German Landrace F2 family with 170 animals. The aim of the present study is to characterize the expression response in this family in order to unravel resistance and susceptibility mechanisms and to prioritize candidate genes for future fine mapping approaches. F2 pigs differed distinctly in clinical, pathological, and microbiological parameters after challenge with A. pleuropneumoniae. We monitored genome-wide gene expression from the 50 most and 50 least susceptible F2 pigs and identified 171 genes differentially expressed between these extreme phenotypes. We combined expression QTL analyses with network analyses and functional characterization using gene set enrichment analysis and identified a functional hotspot on SSC13, including 55 eQTL. The integration of the different results provides a resource for candidate prioritization for fine mapping strategies, such as TF, TFRC, RUNX1, TCN1, HP, CD14, among others.

Identification of QTL affecting resistance/susceptibility to acute Actinobacillus pleuropneumoniae infection in swine.

Actinobacillus pleuropneumoniae is among the most important pathogens worldwide in pig production. The agent can cause severe economic losses due to decreased performance, acute or chronic pleuropneumonia and an increased incidence of death. Therapeutics cannot be used in a sustainable manner, and vaccination is not always available, but discovering more about host defence and disease mechanisms might lead to new methods of prophylaxis. The aim of the present study was to detect quantitative trait loci (QTL) associated with resistance/susceptibility to A. pleuropneumoniae. Under controlled conditions, 170 F2 animals of a Hampshire/Landrace family, with known differences in founder populations regarding A. pleuropneumoniae resistance, were challenged with an A. pleuropneumoniae serotype 7 aerosol followed by a detailed clinical, radiographic, ultrasonographic, pathological and bacteriological examination. F2 pigs were genotyped with 159 microsatellite markers. Significant QTL were identified on Sus scrofa chromosomes (SSC) 2, 6, 12, 13, 16, 17 and 18. They explained 6-22% of phenotypic variance. One QTL on SSC2 reached significance on a genome-wide level for five associated phenotypic traits. A multiple regression analysis revealed a combinatory effect of markers SWR345 (SSC2) and S0143 (SSC12) on Respiratory Health Score, Clinical Score and the occurrence of death. The results indicate the genetic background of A. pleuropneumoniae resistance in swine and provide new insights into the genetic architecture of resistance/susceptibility to porcine pleuropneumonia. The results will be helpful in identifying the underlying genes and mechanisms.

Glycoprotein analysis of porcine bronchoalveolar lavage fluid reveals potential biomarkers corresponding to resistance to Actinobacillus pleuropneumoniae infection.

Biomarkers facilitating both pathogen-independent diagnosis of respiratory health and breeding selection of pigs with increased resistance to respiratory tract infections would be of considerable interest to the pig industry. Following this concept we performed a comparative glycoproteome analysis of bronchoalveolar lavage fluid (BALF) from healthy pigs and pigs 4 days (acute) and 20 days (chronic) after an experimental infection with Actinobacillus pleuropneumoniae. In order to identify possible differences in BALF glycoprotein patterns we investigated pigs of three different breeding lines (German Landrace, Piétrain, Hampshire). In total, 12 glycosylated proteins (alpha-1-acid glycoprotein, fetuin A, properdin, haptoglobin precursor, haptoglobin, hemoglobin, hyaluronidase, inter-alpha-trypsin inhibitor family heavy chain-related protein, alpha-1-antichymotrypsin 3, pulmonary surfactant-associated protein D (SP-D), transferrin, and alpha-1B-glycoprotein) were identified as being differentially expressed depending on the health status of the animal. Fetuin A levels were consistently low in chronically infected pigs thereby being a potential marker for chronic infection. Hyaluronidase levels were consistently high in all pigs after experimental infection independent on isolation of the pathogen thereby being a potential marker for previous pathogen contact and latent infection. High levels of fetuin A as well as low levels of haptoglobin and pulmonary SP-D correlated with the absence of lung lesions in pigs of the Hampshire breeding line, implying a potential application as selection markers for breeding programmes.

[Study of the virulence of Actinobacillus pleuropneumoniae in finishing pigs as a basis for vaccination development]. / Studie zur Virulenz von Actinobacillus pleuropneumoniae bei schweren Mastschweinen als Grundlage für die Entwicklung von Impfstoffen.

For vaccine licensing data about efficiency and duration of protection are essential. Within the scope of the developement of a new subunit vaccine against Actinobacillus pleuropneumoniae (A.pp.) the protective efficiency over the whole length of the fattening period must be proven. This required infection experiments in finishing pigs. Eight pigs in the age of six months were infected experimentally into the trachea with an A.pp. serotyp 2 strain. To our knowledge data about the susceptibility of pigs of this age do not exist, so that the infectious dose for pigs of this age and this route of infection had to be determined. Two pigs each were infected with different doses of 10(10), 6 x 10(5), 8 x 10(3) and 2 x 10(3) CFU (colony forming units). The aim of the study was to produce a typical pleuropneumonia with fever and severe respiratory symptoms as well as characteristic pathomorphological lung alterations without loss of animals during the acute stage of infection. The pathogen should be cultivated from lung tissue. The recommended dose for testing the efficiency of vaccines turned out to be approximately 10(3) CFU A.pp. serotyp 2.

[The implementation of the broth microdilution method to determine bacterial susceptibility to antimicrobial agents]. / Untersuchungen zur Implementierung des Bouillon-mikrodilutionsverfahrens zur Empfindlichkeitsprüfung von Bakterien gegenüber antimikrobiellen Wirkstoffen.

The determination of quantitative data (minimal inhibitory concentrations [MIC values]) using the broth microdilution method is considered the method of choice for in-vitro susceptibility testing of bacterial pathogens. For the inoculation of microtiter plates used in the broth microdilution method, an inoculation density of 5x10(5) CFU/ml has to be achieved according to the CLSI standard. In this study, it was shown that the requested CFU/ml corresponded to an optical density of 0.00027 of a bacterial suspension determined in a spectrophotometer at 625 nm (OD625). The comparison of susceptibility data of clinical isolates determined by agar disk diffusion and, in parallel, by broth microdilution confirmed the comparability of both methods. For most antibiotics the frequency of "very major errors" was below 10%. However, a higher frequency of errors was observed for combinations of antimicrobial agents and bacteria for which no valid breakpoints are available. The comparative testing of cephalothin showed a frequency of"very major errors" of 20.3% for staphylococci and 18.4% for Enterobacteriaceae; for apramycin the frequency of "major errors"was found to be 46.0% for Enterobacteriaceae. The reference strains as recommended in the CLSI Standard M31-S1 should be used for quality assurance.

Differential proteomic analysis reveals increased cathelicidin expression in porcine bronchoalveolar lavage fluid after an Actinobacillus pleuropneumoniae infection.

Accurate definition of respiratory health in pigs is an important problem for swine producers and veterinarians. In an approach to identify potential biomarkers, two-dimensional gel electrophoresis and mass spectrometry on bronchoalveolar lavage fluid (BALF)-derived proteins from pigs experimentally infected with Actinobacillus pleuropneumoniae were performed at different time points post infection. Mock-infected pigs were used as a control. It was shown that the antimicrobial peptides, prophenin-2 and PR-39, and the calcium-binding protein calgranulin C were reproducibly upregulated in BALF of pigs chronically infected with A. pleuropneumoniae. Concentrations of PR-39 were significantly (p<0.05) increased in BALF (median of 4.8 nM) but not in serum (median of 2.5 nM) on day 21 after infection. A Receiver Operating Characteristics (ROC) plot showed that PR-39 in BALF is an accurate and easily accessible marker to detect clinically healthy pigs convalescent from an experimental A. pleuropneumoniae infection. These results imply that PR-39 might have a potential as a general biomarker to determine porcine respiratory health.

Characterization of genetic differences between Mycobacterium avium subsp. paratuberculosis type I and type II isolates.

A combination of representational difference analysis and comparative DNA sequencing revealed that four type I (sheep) isolates of Mycobacterium avium subsp. paratuberculosis were differentiated from nine type II (bovine) isolates by the presence of an 11-bp insertion in a novel M. avium subsp. paratuberculosis-specific region of genomic DNA. Further, our studies show that M. avium subsp. paratuberculosis type I isolates contain three type-specific loci that are missing in M. avium subsp. paratuberculosis type II but are present in M. avium subsp. avium. Taken together, the results are consistent with the hypothesis that M. avium subsp. paratuberculosis type I strains are an evolutionary intermediate between M. avium subsp. avium and M. avium subsp. paratuberculosis type II isolates or share a subset of M. avium subsp. avium type-specific loci through horizontal transfer.