Experimental Actinobacillus pleuropneumoniae challenge in swine: Comparison of computed tomographic and radiographic findings during disease

ABSTRACT: BACKGROUND: In pigs, diseases of the respiratory tract like pleuropneumonia due to Actinobacillus pleuropneumoniae (App) infection have led to high economic losses for decades. Further research on disease pathogenesis, pathogen-host-interactions and new prophylactic and therapeutic approaches are needed. In most studies, a large number of experimental animals are required to assess lung alterations at different stages of the disease. In order to reduce the required number of animals but nevertheless gather information on the nature and extent of lung alterations in living pigs, a computed tomographic scoring system for quantifying gross pathological findings was developed. In this study, five healthy pigs served as control animals while 24 pigs were infected with App, the causative agent of pleuropneumonia in pigs, in an established model for respiratory tract disease. RESULTS: Computed tomographic (CT) findings during the course of App challenge were verified by radiological imaging, clinical, serological, gross pathology and histological examinations. Findings from clinical examinations and both CT and radiological imaging, were recorded on day 7 and day 21 after challenge. Clinical signs after experimental App challenge were indicative of acute to chronic disease. Lung CT findings of infected pigs comprised ground-glass opacities and consolidation. On day 7 and 21 the clinical scores significantly correlated with the scores of both imaging techniques. At day 21, significant correlations were found between clinical scores, CT scores and lung lesion scores. In 19 out of 22 challenged pigs the determined disease grades (not affected, slightly affected, moderately affected, severely affected) from CT and gross pathological examination were in accordance. Disease classification by radiography and gross pathology agreed in 11 out of 24 pigs. CONCLUSIONS: High-resolution, high-contrast CT examination with no overlapping of organs is superior to radiography in the assessment of pneumonic lung lesions after App challenge. The new CT scoring system allows for quantification of gross pathological lung alterations in living pigs. However, computed tomographic findings are not informative of the etiology of respiratory disease.     

Peptide aMptD-Mediated Capture PCR for Detection of Mycobacterium avium subsp. paratuberculosis in Bulk Milk Samples

A peptide-mediated capture PCR for the detection of Mycobacterium avium subsp. paratuberculosis in bulk milk samples was developed and characterized. Capture of the organism was performed using peptide aMptD, which had been shown to bind to the M. avium subsp. paratuberculosis MptD protein (J. Stratmann, B. Strommenger, R. Goethe, K. Dohmann, G. F. Gerlach, K. Stevenson, L. L. Li, Q. Zhang, V. Kapur, and T. J. Bull, Infect. Immun. 72:1265-1274, 2004). Consistent expression of the MptD receptor protein and binding of the aMptD ligand were demonstrated by capturing different Mycobacterium avium subsp. paratuberculosis type I and type II strains and subsequent PCR analysis using ISMav2-based primers. The analytical sensitivity of the method was determined to be 5 × 10² CFU ml⁻¹ for artificially contaminated milk. The specificity of aMptD binding was confirmed by culture and competitive capture assays, showing selective enrichment of M. avium subsp. paratuberculosis (at a concentration of 5 × 10² CFU ml⁻¹) from samples containing 100- and 1,000-fold excesses of other mycobacterial species, including M. avium subsp. avium and M. avium subsp. hominissuis. The aMptD-mediated capture of M. avium subsp. paratuberculosis using paramagnetic beads, followed by culture, demonstrated the ability of this approach to capture viable target cells present in artificially contaminated milk. Surface plasmon resonance experiments revealed that the aMptD peptide is a high-affinity ligand with a calculated association rate constant of 9.28 × 10³ and an association constant of 1.33 × 10⁹. The potential use of the method on untreated raw milk in the field was investigated by testing 423 bulk milk samples obtained from different dairy farms in Germany, 23 of which tested positive. Taken together, the results imply that the peptide-mediated capture PCR might present a suitable test for paratuberculosis screening of dairy herds, as it has an analytical sensitivity sufficient for detection of M. avium subsp. paratuberculosis in bulk milk samples under field conditions, relies on a defined and validated ligand-receptor interaction, and is adaptable to routine diagnostic laboratory automation.     

Peptide aMptD-mediated capture PCR for detection of Mycobacterium avium subsp. paratuberculosis in bulk milk samples

A peptide-mediated capture PCR for the detection of Mycobacterium avium subsp. paratuberculosis in bulk milk samples was developed and characterized. Capture of the organism was performed using peptide aMptD, which had been shown to bind to the M. avium subsp. paratuberculosis MptD protein (J. Stratmann, B. Strommenger, R. Goethe, K. Dohmann, G. F. Gerlach, K. Stevenson, L. L. Li, Q. Zhang, V. Kapur, and T. J. Bull, Infect. Immun. 72:1265-1274, 2004). Consistent expression of the MptD receptor protein and binding of the aMptD ligand were demonstrated by capturing different Mycobacterium avium subsp. paratuberculosis type I and type II strains and subsequent PCR analysis using ISMav2-based primers. The analytical sensitivity of the method was determined to be 5 x 10(2) CFU ml(-1) for artificially contaminated milk. The specificity of aMptD binding was confirmed by culture and competitive capture assays, showing selective enrichment of M. avium subsp. paratuberculosis (at a concentration of 5 x 10(2) CFU ml(-1)) from samples containing 100- and 1,000-fold excesses of other mycobacterial species, including M. avium subsp. avium and M. avium subsp. hominissuis. The aMptD-mediated capture of M. avium subsp. paratuberculosis using paramagnetic beads, followed by culture, demonstrated the ability of this approach to capture viable target cells present in artificially contaminated milk. Surface plasmon resonance experiments revealed that the aMptD peptide is a high-affinity ligand with a calculated association rate constant of 9.28 x 10(3) and an association constant of 1.33 x 10(9). The potential use of the method on untreated raw milk in the field was investigated by testing 423 bulk milk samples obtained from different dairy farms in Germany, 23 of which tested positive. Taken together, the results imply that the peptide-mediated capture PCR might present a suitable test for paratuberculosis screening of dairy herds, as it has an analytical sensitivity sufficient for detection of M. avium subsp. paratuberculosis in bulk milk samples under field conditions, relies on a defined and validated ligand-receptor interaction, and is adaptable to routine diagnostic laboratory automation.     

Characterization of the genes encoding urease activity of Klebsiella pneumoniae

Abstract The genes encoding urease activity of Klebsiella pneumoniae were cloned and expressed in Escherichia coli . Transformants containing recombinant plasmids were selected by the antibiotic resistance phenotype and the production of urease in a Urease-test agar. Deletion derivatives of the parental recombinant plasmid were construced, and the relative position of six genes, necessary for urease production, was determined. Using a colorimetric assay it was demonstrated that some of the transformants exhibited ureolytic activity up to six-times greater than that of the original K pneumoniae isolate. Dot-blot DNA hybridization analysis revealed that the urease gene cluster of K. pneumoniae possesses no significant homology with those of Proteus species and Morganella morganii .     

A single-step transconjugation system for the introduction of unmarked deletions into Actinobacillus pleuropneumoniae serotype 7 using a sucrose sensitivity marker

Research on the porcine respiratory tract pathogen Actinobacillus pleuropneumoniae requires the availability of improved genetic tools. Therefore, using the sacB gene of Bacillus subtilis, we developed a sucrose-based counterselection system that allows rapid curing of an Escherichia coli-A. pleuropneumoniae shuttle vector as well as the introduction of unmarked mutations into the A. pleuropneumoniae chromosome. A cassette containing the Tn903 kanamycin resistance determinant (km(r)) and the sacB gene expressed from the A. pleuropneumoniae omlA promoter was introduced by homologous recombination into the ureC gene of A. pleuropneumoniae. The resultant stable plasmid cointegrates were kanamycin-resistant, sucrose-sensitive, and urease-positive. A simple counterselection on sucrose-containing agar plates without an additional transconjugation step allowed the efficient isolation of urease-negative A. pleuropneumoniae mutants that had lost the km(r)-sacB cassette.     

Identification of Brachyspira hyodysenteriae-specific DNA fragments using representational difference analysis

Two novel Brachyspira hyodysenteriae-specific DNA fragments, designated as Bh100 and Bh400, were identified using representational difference analysis. To isolate the fragments the combined DNA of the Brachyspira pilosicoli, Brachyspira intermedia, Brachyspira murdochii and Brachyspira innocens reference strains was subtracted from the genome of B. hyodysenteriae strain B204. Both fragments were present in a single copy and mapped to different positions on the genome of B. hyodysenteriae B78(T). Larger fragments encompassing the continuous open reading frames (ORF) of Bh100 and Bh400 were cloned and analysed. Whereas the ORF of 2130 bp encompassing Bh100 did not show homology to any known bacterial protein, Bh400 was part of a putative operon with significant homology to the phosphotransferase system of Bacillus subtilis.     

First Chromosomal Restriction Map of Actinobacillus pleuropneumoniae and Localization of Putative Virulence-Associated Genes

Combined physical and genetic maps of the genomes of Actinobacillus pleuropneumoniae AP76 (serotype 7 clinical isolate) and of A. pleuropneumoniae ATCC 27088 (serotype 1 reference strain) were constructed by using the restriction endonucleases ApaI, AscI, NotI, and SalI. The chromosome sizes as determined by the addition of estimated fragment sizes were 2.4 Mbp, and both maps had a resolution of approximately 100 kbp. The linkages between the ApaI, AscI, NotI, and SalI fragments and their relative positions were determined by (i) fragment excision and redigestion and (ii) partial digests of defined fragments and Southern blot using end-standing probes. The single SalI site within the chromosome of strain A. pleuropneumoniae AP76 was defined as position 1 of the map; for the map of A. pleuropneumoniae ATCC 27088, the corresponding SalI site was chosen. Putative virulence-associated genes (apx, omlA, sodA, tbpBA, ureC, and a repeat element) and housekeeping genes (glyA, metJ, recA, and rhoAP) were positioned on the physical maps and located on the ApaI and NotI fragments of A. pleuropneumoniae serotype reference strains.     

Cloning and characterization of the gene cluster encoding type 3 (MR/K) fimbriae of Klebsiella pneumoniae

Abstract The gene cluster encoding the type 3 fimbriae of a Klebsiella pneumoniae isolate was cloned using the cosmid-cloning technique. Escherichia coli transformants, expressing type 3 fimbriae, were selected by reactivity with a monoclonal antibody directed against an epitope of the purified type 3 fimbriae. The phenotypic expression of type 3 fimbriae by transformants possessing the parental plasmid was dependent upon the host strain used. However, subcloning of this plasmid resulted in the construction of a chimeric molecule which imparted a stable phenotype regardless of the host strain. In addition, subcloning of the parental recombinant plasmid suggested that the minimal size of DNA necessary for production and expression of fimbriae was approximately 5.5 kb.     

Enrichment of open reading frames presented on bacteriophage M13 using hyperphage

The enrichment of open reading frames (ORFs) from large gene libraries and the presentation of the corresponding polypeptides on filamentous phage M13 (phage display) is frequently used to identify binding partners of unknown ORFs. In particular phage display is a valuable tool for the identification of pathogen-related antigens and a first step for the development of new diagnostics and therapeutics. Here, we introduce a significant improvement of phage-based ORF enrichment by using Hyperphage, a helperphage with a truncated gIII. The methods allow both the enrichment of ORFs from cDNA libraries and the display of the corresponding polypeptides on phage, thus combining ORF enrichment with a screening for binding in one step without any further subcloning steps. We demonstrated the benefits of the method by isolating the sequences encoding two predicted immunogenic epitopes of the outer membrane protein D encoding gene (ompD) of Salmonella typhimurium. Here, we showed that when using a mixture of three constructs with only one containing an ORF solely this correct construct could be reisolated in phage particles. Further; both epitopes were detected by enzyme-linked immunosorbent assay (ELISA), demonstrating correct translation of fusion proteins. Furthermore, the enrichment system was evaluated by the enrichment of ORFs from total cDNA of lymphocytes. Here, we could show that 60% of the phage contained ORFs, which is an increase of an order of magnitude compared with conventional phage expression system. Together these data show that the Hyperphage-based enrichment system significantly improves the enrichment of ORFs and directly allows the display of the corresponding polypeptide on bacteriophage M13.