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ABSTRACTZoonotic infections caused by Salmonella entericaserovar Typhimurium pose a constant threat to consumer health, with the pig being a particularly major source of multidrug-resistant isolates. Vaccination, as a promising approach to reduce colonization and shedding, has been scarcely used, as it interferes with current control programs relying on serology as a means of herd classification. In order to overcome this problem, we set out to develop a negative-marker vaccine allowing the differentiation of infected from vaccinated animals (DIVA). Applying an immunoproteomic approach with two-dimensional gel electrophoresis, Western blot, and quadrupole time-of-flight tandem mass spectrometry, we identified the OmpD protein as a suitable negative marker. Using allelic exchange, we generated an isogenic mutant of the licensed live vaccine strain Salmoporc and showed that virulence of Salmoporc and that of the mutant strain, SalmoporcΔompD, were indistinguishable in BALB/c mice. In a pig infection experiment including two oral immunizations with SalmoporcΔompDand challenge with a multiresistant S. entericaserovar Typhimurium DT104 clinical isolate, we confirmed the protective efficacy of SalmoporcΔompDin pigs, showing a significant reduction of both clinical symptoms and colonization of lymph nodes and intestinal tract. OmpD immunogenic epitopes were determined by peptide spot array analyses. Upon testing of several 9-mer peptides, each including an immunogenic epitope, one peptide (positions F100to Y108) that facilitated the detection of infected animals independent of their vaccination status (DIVA function) was identified. The approach described overcomes the problems currently limiting the use of bacterial live vaccines and holds considerable potential for future developments in the field.

ABSTRACTVaccination against Actinobacillus pleuropneumoniaeis hampered by the lack of vaccines inducing reliable cross-serotype protection. In contrast, pigs surviving natural infection are at least partially protected from clinical symptoms upon reinfection with any serotype. Thus, we set out to construct an attenuated A. pleuropneumoniaelive vaccine allowing the differentiation of vaccinated from infected animals (the DIVA concept) by successively deleting virulence-associated genes. Based on an A. pleuropneumoniaeserotype 2 prototype live negative marker vaccine (W. Tonpitak, N. Baltes, I. Hennig-Pauka, and G.-F. Gerlach, Infect. Immun. 70:7120-7125, 2002), genes encoding three enzymes involved in anaerobic respiration and the ferric uptake regulator Fur were deleted, resulting in a highly attenuated sixfold mutant; this mutant was still able to colonize the lower respiratory tract and induced a detectable immune response. Upon a single aerosol application, this mutant provided significant protection from clinical symptoms upon heterologous infection with an antigenically distinct A. pleuropneumoniaeserotype 9 challenge strain and allowed the serological discrimination between infected and vaccinated groups.

ABSTRACTActinobacillus pleuropneumoniae, the etiological agent of porcine pleuropneumonia, is able to persist on respiratory epithelia, in tonsils, and in the anaerobic environment of encapsulated lung sequesters. We have demonstrated previously that putative HlyX-regulated genes, coding for dimethyl sulfoxide (DMSO) reductase and aspartate ammonia lyase, are upregulated during infection and that deletions in these genes result in attenuation of the organism. The study presented here investigates the role of HlyX, the fumarate nitrate reductase regulator (FNR) homologue of A. pleuropneumoniae. By constructing an isogenic A. pleuropneumoniae hlyXmutant, the HlyX protein is shown to be responsible for upregulated expression of both DMSO reductase and aspartate ammonia lyase (AspA) under anaerobic conditions. In a challenge experiment the A. pleuropneumoniae hlyXmutant is shown to be highly attenuated, unable to persist in healthy lung epithelium and tonsils, and impaired in survival inside sequestered lung tissue. Further, using an A. pleuropneumoniaestrain carrying the luxABgenes as transcriptional fusion to aspAon the chromosome, the airway antioxidant glutathione was identified as one factor potentially responsible for inducing HlyX-dependent gene expression of A. pleuropneumoniaein epithelial lining fluid.

ABSTRACTGenes expressed by Actinobacillus pleuropneumoniaein necrotic porcine lung tissue were identified by selective capture of transcribed sequences analysis. In total, 46 genes were identified, 20 of which have been previously reported to be associated with in vivo expression or virulence in A. pleuropneumoniaeor in other organisms.

We have used representational difference analysis to identify a novel Mycobacterium avium subsp. paratuberculosis-specific ABC transporter operon (mpt), which comprises six open reading frames designated mptA to -F and is immediately preceded by two putative Fur boxes. Functional genomics revealed that the mpt operon is flanked on one end by a fep cluster encoding proteins involved in the uptake of Fe(3+) and on the other end by a sid cluster encoding non-ribosome-dependent heterocyclic siderophore synthases. Together these genes form a 38-kb M. avium subsp. paratuberculosis-specific locus flanked by an insertion sequence similar to IS1110. Expression studies using Western blot analyses showed that MptC is present in the envelope fraction of M. avium subsp. paratuberculosis. The MptD protein was shown to be surface exposed, using a specific phage (fMptD) isolated from a phage-peptide library, by differential screening of Mycobacterium smegmatis transformants. The phage fMptD-derived peptide could be used in a peptide-mediated capture PCR with milk from infected dairy herds, thereby showing surface-exposed expression of the MptD protein in the host. Together, these data suggest that the 38-kb locus constitutes an M. avium subsp. paratuberculosis pathogenicity island.

ABSTRACTWe have used representational difference analysis to identify a novel Mycobacterium aviumsubsp. paratuberculosis-specific ABC transporter operon (mpt), which comprises six open reading frames designated mptAto -Fand is immediately preceded by two putative Fur boxes. Functional genomics revealed that the mptoperon is flanked on one end by a fepcluster encoding proteins involved in the uptake of Fe3+and on the other end by a sidcluster encoding non-ribosome-dependent heterocyclic siderophore synthases. Together these genes form a 38-kb M. aviumsubsp. paratuberculosis-specific locus flanked by an insertion sequence similar to IS1110. Expression studies using Western blot analyses showed that MptC is present in the envelope fraction of M. aviumsubsp. paratuberculosis. The MptD protein was shown to be surface exposed, using a specific phage (fMptD) isolated from a phage-peptide library, by differential screening of Mycobacterium smegmatistransformants. The phage fMptD-derived peptide could be used in a peptide-mediated capture PCR with milk from infected dairy herds, thereby showing surface-exposed expression of the MptD protein in the host. Together, these data suggest that the 38-kb locus constitutes an M. aviumsubsp. paratuberculosispathogenicity island.

Actinobacillus pleuropneumoniae, the causative agent of porcine pleuropneumonia, is capable of persisting in oxygen-deprived surroundings, namely, tonsils and sequestered necrotic lung tissue. Utilization of alternative terminal electron acceptors in the absence of oxygen is a common strategy in bacteria under anaerobic growth conditions. In an experiment aimed at identification of genes expressed in vivo, the putative catalytic subunit DmsA of anaerobic dimethyl sulfoxide reductase was identified in an A. pleuropneumoniae serotype 7 strain. The 90-kDa protein exhibits 85% identity to the putative DmsA protein of Haemophilus influenzae, and its expression was found to be upregulated under anaerobic conditions. Analysis of the unfinished A. pleuropneumoniae genome sequence revealed putative open reading frames (ORFs) encoding DmsB and DmsC proteins situated downstream of the dmsA ORF. In order to investigate the role of the A. pleuropneumoniae DmsA protein in virulence, an isogenic deletion mutant, A. pleuropneumoniae DeltadmsA, was constructed and examined in an aerosol infection model. A. pleuropneumoniae DeltadmsA was attenuated in acute disease, which suggests that genes involved in oxidative metabolism under anaerobic conditions might contribute significantly to A. pleuropneumoniae virulence.

ABSTRACTActinobacillus pleuropneumoniae, the causative agent of porcine pleuropneumonia, is capable of persisting in oxygen-deprived surroundings, namely, tonsils and sequestered necrotic lung tissue. Utilization of alternative terminal electron acceptors in the absence of oxygen is a common strategy in bacteria under anaerobic growth conditions. In an experiment aimed at identification of genes expressed in vivo, the putative catalytic subunit DmsA of anaerobic dimethyl sulfoxide reductase was identified in an A. pleuropneumoniaeserotype 7 strain. The 90-kDa protein exhibits 85% identity to the putative DmsA protein of Haemophilus influenzae, and its expression was found to be upregulated under anaerobic conditions. Analysis of the unfinished A. pleuropneumoniaegenome sequence revealed putative open reading frames (ORFs) encoding DmsB and DmsC proteins situated downstream of the dmsAORF. In order to investigate the role of the A. pleuropneumoniaeDmsA protein in virulence, an isogenic deletion mutant, A. pleuropneumoniaeΔdmsA, was constructed and examined in an aerosol infection model. A. pleuropneumoniaeΔdmsAwas attenuated in acute disease, which suggests that genes involved in oxidative metabolism under anaerobic conditions might contribute significantly to A. pleuropneumoniaevirulence.

ABSTRACTDeletions were introduced into the ureCand apxIIAgenes of an Actinobacillus pleuropneumoniaeserotype 2 strain by homologous recombination and counterselection. The double-mutant contains no foreign DNA, is highly attenuated, protects pigs from homologous challenge upon a single aerosol application, and facilitates the serological discrimination of immunized and infected herds.

ABSTRACTActinobacillus pleuropneumoniae, a porcine respiratory tract pathogen, has been shown to express transferrin-binding proteins and urease during infection. Both activities have been associated with virulence; however, their functional role for infection has not yet been elucidated. We used two isogenic A. pleuropneumoniaesingle mutants (ΔexbBand ΔureC) and a newly constructed A. pleuropneumoniaedouble (ΔureCΔexbB) mutant in aerosol infection experiments. Neither the A. pleuropneumoniaeΔexbBmutant nor the double ΔureCΔexbBmutant was able to colonize sufficiently long to initiate a detectable humoral immune response. These results imply that the ability to utilize transferrin-bound iron is required for multiplication and persistence of A. pleuropneumoniaein the porcine respiratory tract. TheA. pleuropneumoniaeΔureCmutant and the parent strain both caused infections that were indistinguishable from one another in the acute phase of disease; however, 3 weeks postinfection the A. pleuropneumoniaeΔureCmutant, in contrast to the parent strain, could not be isolated from healthy lung tissue. In addition, the local immune response—as assessed by fluorescence-activated cell sorter and enzyme-linked immunosorbent spot analyses—revealed a significantly higher number ofA. pleuropneumoniae-specific B cells in the bronchoalveolar lavage fluid (BALF) of pigs infected with the A. pleuropneumoniaeΔureCmutant than in the BALF of those infected with the parent strain. These results imply thatA. pleuropneumoniaeurease activity may cause sufficient impairment of the local immune response to slightly improve the persistence of the urease-positive A. pleuropneumoniaeparent strain.

ABSTRACTUpon iron restriction, Actinobacillus pleuropneumoniaehas been shown to express the transferrin-binding proteins TbpB and TbpA, both of which have been implied to be important virulence factors. In order to identify additional iron-regulated proteins, we cloned and analyzed the region upstream of the transferrin-binding protein genes in an A. pleuropneumoniaeserotype 7 strain. We located immediately upstream of the tbpBgene two open reading frames which were 43% homologous to the neisserial ExbBD protein genes. By raising specific antibodies, we showed that ExbB is expressed under iron-limiting growth conditions only, and RT-PCR analysis revealed that the exbBDgenes and thetbpBgene are transcribed on a single polycistronic mRNA. By constructing an isogenic and nonpolar exbBDmutant, we showed that the exbBDgenes are required by A. pleuropneumoniaefor utilization of transferrin-bound iron. Using PCR and Western blotting, we showed that the genetic organization found in A. pleuropneumoniaeserotype 7 is similar in all 12A. pleuropneumoniaeserotype reference strains.