Draft Genome Sequence of Actinobacillus pleuropneumoniae Serotype 7 Strain S-8

The Gram-negative bacterium Actinobacillus pleuropneumoniae is the etiological agent of porcine pleuropneumonia, a respiratory disease that leads to severe economic losses in the swine industry. For years, scientists working with it have lacked a reliable genome sequence for comparison with other Actinobacillus species. Here, we report the draft genome sequence of A. pleuropneumoniae serotype 7 (strain S-8), isolated from swine lung in China in 1992.     

Evaluation of PCR Based on Gene <Emphasis Type="Italic">apxIVA</Emphasis> Associated with 16S rDNA Sequencing for the Identification of <Emphasis Type="Italic">Actinobacillus pleuropneumoniae</Emphasis> and Related Species

The pleuropneumonia caused by Actinobacillus pleuropneumoniae (App) is one the most important swine respiratory diseases. Biochemical and serological tests are widely applied for App diagnosis and characterization. However, in some isolates, conflicting results are found. The present work focus on the characterization of 29 isolates biochemically classified as A. pleuropneumoniae, collected from swine in herds with or without a clinical history of pleuropneumonia. Sixteen isolates were from healthy swine, initially classified as nonserotypable A. pleuropneumoniae; they displayed differences in the molecular characterization patterns of App (genes cpx and apxI, II, and III). Those bacteria that could not be serotyped were submitted to rDNA 16S sequencing. All 29 isolates were analyzed by PCR for the presence of the apxIVA gene. Thirteen isolates (45%) were confirmed to be A. pleuropneumoniae by PCR, nine being from diseased animals (31%) and four from healthy animals (14%) with conclusive serotyping. The rDNA 16S sequencing was used to classify the other 16 isolates in related species other than A. pleuropneumoniae, resulting in eleven A. minor, three A. porcinus, and two Pasteurella sp. Because of conflicting results between biochemical tests and rDNA 16S sequencing, the biochemical characterization was repeated, and the new results were in agreement with the rDNA 16S sequencing data. Biochemical characterization proved to be efficient for the majority of the A. pleuropneumoniae isolates. Nevertheless, conventional tests can render conflicting results, and other methodologies, such as amplification of A. pleuropneumoniae specific apxIVA gene and rDNA 16S sequencing, are very useful for improved classification. We also observed a great variety in rDNA 16S sequences from different A. minor isolates.     

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.     

Detection of <Emphasis Type="Italic">Actinobacillus pleuropneumoniae</Emphasis> by PCR on Field Strains from Healthy and Diseased Pigs

We investigated whether primers able to specifically amplify a 0.7-kb DNA fragment from the conserved cpx genes could be applied to analyze A. pleuropneumoniae field isolates. The specific cpx primers were tested on 120 strains of A. pleuropneumoniae and other NAD-dependent field isolates from healthy and diseased animals to analyze A. pleuropneumoniae isolates from pigs in Brazil. We found that PCR and hybridization were able to discriminate between isolates of A. pleuropneumoniae and other bacteria. The 0.7-kb cpx DNA fragments were amplified from all 63 A. pleuropneumoniae isolates from herds with clinical symptoms and were isolated from lesions of acute cases of swine pleuropneumonia, both serotypable and nonserotypable. The PCR was also applied to 57 field isolates obtained from animals of apparently healthy herds, and the amplified cpx product was present in four serotypable and only two out of eleven A. pleuropneumoniae nonserotypable isolates. All nonserotypable A. pleuropneumoniae isolates revealed the apxA amplification pattern compatible with previously known serotypes. Some nonserotypable isolates might represent a population of isolates that originally were serotypable but lost the ability to react with serotype-specific antisera or might belong to novel serotypes. The PCR method applied is highly sensitive for serotypable A. pleuropneumoniae strains and for nonserotypable strains isolated from acute cases of swine pleuropneumoniae in Brazil. Received: 13 June 2002 / Accepted: 5 August 2002     

Comparative Genomic Characterization of Actinobacillus pleuropneumoniae

The Gram-negative bacterium Actinobacillus pleuropneumoniae is the etiologic agent of porcine contagious pleuropneumoniae, a lethal respiratory infectious disease causing great economic losses in the swine industry worldwide. In order to better interpret the genetic background of serotypic diversity, nine genomes of A. pleuropneumoniae reference strains of serovars 1, 2, 4, 6, 9, 10, 11, 12, and 13 were sequenced by using rapid high-throughput approach. Based on 12 genomes of corresponding serovar reference strains including three publicly available complete genomes (serovars 3, 5b, and 7) of this bacterium, we performed a comprehensive analysis of comparative genomics and first reported a global genomic characterization for this pathogen. Clustering of 26,012 predicted protein-coding genes showed that the pan genome of A. pleuropneumoniae consists of 3,303 gene clusters, which contain 1,709 core genome genes, 822 distributed genes, and 772 strain-specific genes. The genome components involved in the biogenesis of capsular polysaccharide and lipopolysaccharide O antigen relative to serovar diversity were compared, and their genetic diversity was depicted. Our findings shed more light on genomic features associated with serovar diversity of A. pleuropneumoniae and provide broader insight into both pathogenesis research and clinical/epidemiological application against the severe disease caused by this swine pathogen.Actinobacillus pleuropneumoniae, a Gram-negative facultative anaerobic encapsulated coccobacillus, belongs to the Actinobacillus genus of the Pasteurellaceae family (19). A. pleuropneumoniae is a primary bacterial etiologic agent of porcine contagious pleuropneumonia, a severe respiratory disease leading to great economic losses to the global swine industry (7). The cases usually display pleuropneumonia and pulmonary lesions characterized by serious hemorrhage and necrosis. To date, several factors involved in the virulence of A. pleuropneumoniae have been described, including Apx exotoxins, capsular polysaccharides (CPS), lipopolysaccharides (LPS), outer membrane proteins, iron-acquisition proteins and adhesin factors (11, 19, 24). However, the genetic differences of pathogenesis remain poorly characterized and are worth interpreting from the perspective of comparative genomics for this bacterium.Thus far, 15 serovars and two biotypes of A. pleuropneumoniae have been recognized, with great variations in virulence and interlocal distributions (6). The predominant serovar-specific antigens are composed of CPS, which could rigorously define serovars of A. pleuropneumoniae (6, 34). Antigenic differences in the LPS can further determine A. pleuropneumoniae subtypes within a same capsular serovar (13). The metabolic and virulent characteristics of this pathogen have been systematically described based on the prior knowledge and two complete genomes (18, 47), but the molecular basis and evolutionary mechanism of serotypic diversity are still not well explained due to the lack of sequence information. To investigate the associations of serovar diversity with the underlying genetic components, more serovar-related genomic islands involved in the biosynthesis of capsular and lipopolysaccharide antigens should be decoded at the pan-genome level of A. pleuropneumoniae. At present, through the next-generation of sequencing technique (454 GS FLX pyrosequencing platform), more and more bacterial species, subspecies or typical strains have been quickly sequenced, such as eight species in the Yersinia genus (9), 17 strains of Streptococcus pneumoniae (22), and 5 strains from different Francisella tularensis subspecies (8). Multiple genome sequences from different strains of a single species can offer comprehensive information to explore the relationship between genotypes and phenotypes and to further discover additional genetic markers for clinical purpose.In the present study, we sequenced the A. pleuropneumoniae genomes of nine reference strains of serovars 1, 2, 4, 6, 9, 10, 11, 12, and 13. Together with three public complete genome sequences of A. pleuropneumoniae serovars 3, 5b, and 7, the analysis of comparative genomics was performed to report a global genomic characterization of this pathogenic bacterium. The acquisition and loss of genome compositions that contribute to virulence and serovar diversity were identified. The genetic loci involved in the biogenesis of capsule and O-specific polysaccharide were compared, and their vital roles in serotypic diversity were investigated.     

Screening of Actinobacillus pleuropneumoniae LuxS Inhibitors

LuxS, a conserved bacterial enzyme involved in the activated methyl cycle, catalyzes S-ribosylhomocysteine (SRH) into homocysteine and AI-2 (the inter-species quorum-sensing signal molecule). This enzyme has been reported to be essential for the survival of Actinobacillus pleuropneumoniae in its natural host. Therefore, it is a potential drug target against A. pleuropneumoniae, an important swine respiratory pathogen causing great economic losses in the pig industry worldwide. In this study, the enzymatic activity determination method was established using the recombinant LuxS of A. pleuropneumoniae. Thirty-five compounds similar to the shape of SRH were screened from the Specs compound library by the software vROCS and were evaluated for LuxS inhibition. Three compounds could inhibit LuxS activity. Two of them were confirmed to be competitive inhibitors and the third one was uncompetitive. All the three compounds displayed inhibitory effects on the growth of A. pleuropneumoniae and two other important swine pathogens, Haemophilis parasuis and Streptococcus suis, with MIC₅₀ values ranging from 11 to 51 μg/ml. No significant cytotoxic effect of the compounds was detected on porcine PK-15 cells at the concentration which showed inhibitory effect on bacterial growth. These results suggest that LuxS is an ideal target to develop antimicrobials for porcine bacterial pathogens. The three LuxS inhibitors identified in this study can be used as lead compounds for drug design.     

Identification of conserved surface proteins as novel antigenic vaccine candidates of Actinobacillus pleuropneumoniae

Actinobacillus pleuropneumoniae is an important swine respiratory pathogen causing great economic losses worldwide. Identification of conserved surface antigenic proteins is helpful for developing effective vaccines. In this study, a genome-wide strategy combined with bioinformatic and experimental approaches, was applied to discover and characterize surface-associated immunogenic proteins of A. pleuropneumoniae. Thirty nine genes encoding outer membrane proteins (OMPs) and lipoproteins were identified by comparative genomics and gene expression profiling as being-highly conserved and stably transcribed in the different serotypes of A. pleuropneumoniae reference strains. Twelve of these conserved proteins were successfully expressed in Escherichia coli and their immunogenicity was estimated by homologous challenge in the mouse model, and then three of these proteins (APJL_0126, HbpA and OmpW) were further tested in the natural host (swine) by homologous and heterologous challenges. The results showed that these proteins could induce high titers of antibodies, but vaccination with each protein individually elicited low protective immunity against A. pleuropneumoniae. This study gives novel insights into immunogenicity of the conserved OMPs and lipoproteins of A. pleuropneumoniae. Although none of the surface proteins characterized in this study could individually induce effective protective immunity against A. pleuropneumoniae, they are potential candidates for subunit vaccines in combination with Apx toxins.     

Characterization of the omlA gene from different serotypes of Actinobacillus pleuropneumoniae: A new insight into an old approach

The OmlA protein is a virulence factor of Actinobacillus pleuropneumoniae, an important pathogen in pigs. The polymorphisms present in the omlA gene sequence of 15 reference serotypes of A. pleuropneumoniae and non-serotypable isolates were assessed to determine the possible evolutionary relationship among them and to validate the importance of this gene as a molecular marker for the characterization of this bacterium. Divergence among the 15 serotypes of A. pleuropneumoniae probably resulted initially from two major evolutionary events that led to subsequent differentiation into nine groups. This differentiation makes it possible to characterize most of the serotypes by using bionformatics, thereby avoiding problems with immunological cross-reactivity. A conserved α-helix common to all the serotypes was most likely involved in connecting the protein to the outer membrane and acting as a signal peptide. A previously unknown gene duplication was also identified and could contribute to the genetic variability that makes it difficult to serotype some isolates. Our data support the importance of the omlA gene in the biology of A. pleuropneumoniae and provide a new area of research into the OmlA protein.     

Multiplex PCR assay for detection of <Emphasis Type="Italic">Actinobacillus pleuropneumoniae,Pasteurella multocida</Emphasis> and <Emphasis Type="Italic">Haemophilus parasuis</Emphasis> in lungs of pigs from a slaughterhouse

Multiplex PCR has been developed for parallel identification of Actinobacillus pleuropneumoniae, Pasteurella multocida and Haemophilus parasuis, important pathogens of swine, responsible for considerable economic losses in swine industry. Multiplex PCR and bacteriological cultivation was used to analyze lung samples from slaughterhouse pigs. From a total of 219 lung samples, 164 (74.9 %) were positive for P. multocida, 45 (20.5 %) for A. pleuropneumoniae and 4 (1.83 %) for H. parasuis. Bacteriological examination revealed that 145 samples (66.2 %) were positive for P. multocida, 31 (14.2 %) for A. pleuropneumoniae and 2 (0.91 %) for H. parasuis.     

Identification of drug target candidates of the swine pathogen <Emphasis Type="Italic">Actinobacillus pleuropneumoniae</Emphasis> by construction of protein–protein interaction network

Porcine pleuropneumonia caused by Actinobacillus pleuropneumoniae has led to severe economic losses in the pig industry worldwide. A. pleuropneumoniae displays various levels of antimicrobial resistance, leading to the dire need to identify new drug targets. Protein–protein interaction (PPI) network can aid the identification of drug targets by discovering essential proteins during the life of bacteria. The aim of this study is to identify drug target candidates of A. pleuropneumoniae from essential proteins in PPI network. The homologous protein mapping method (HPM) was utilized to construct A. pleuropneumoniae PPI network. Afterwards, the subnetwork centered with H-NS was selected to verify the PPI network using bacterial two-hybrid assays. Drug target candidates were identified from the hub proteins by analyzing the topology of the network using interaction degree and homologous comparison with the pig proteome. An A. pleuropneumoniae PPI network containing 2737 non-redundant interaction pairs among 533 proteins was constructed. These proteins were distributed in 21 COG functional categories and 28 KEGG metabolic pathways. The A. pleuropneumoniae PPI network was scale free and the similar topological tendencies were found when compared with other bacteria PPI network. Furthermore, 56.3% of the H-NS subnetwork interactions were validated. 57 highly connected proteins (hub proteins) were identified from the A. pleuropneumoniae PPI network. Finally, 9 potential drug targets were identified from the hub proteins, with no homologs in swine. This study provides drug target candidates, which are promising for further investigations to explore lead compounds against A. pleuropneumoniae.     

In vitro activity of the lipopeptide PAL-Lys-Lys-NH2, alone and in combination with antifungal agents, against clinical isolates of Candida spp

Candida albicans is known to be the organism most often associated with serious fungal infection, but other Candida spp. are emerging as clinical pathogens associated with opportunistic infections. Among antimycotic treatments, increasing attention is currently given to anti-infective drugs based upon naturally occurring peptides, such as the short lipopeptide palmitoyl PAL-Lys-Lys-NH2 (PAL). The aim of this study is to evaluate the activity of this peptide compared to the traditional antifungal agents Fluconazole (FLU), amphotericin B (AMB) and caspofungin (CAS) on Candida spp. 24 clinical isolates of Candida spp. were tested against PAL_, FLU, AMB and CAS using in vitro susceptibility tests, time killing and checkerboard assay. All of the drugs studied showed good activity against clinical isolates of candida; in particular CAS and AMB which have MICs value lower than PAL and FLU. Moreover we observed synergistic interactions for PAL/FLU (81.25%), PAL/AMB (75%) and particularly for PAL/CAS (87.5). We think that our results are interesting since synergy between PAL and CAS might be useful in clinic trails to treat invasive fungal infections.     

Bird species richness and abundance in riparian vegetation invaded by exotic Reynoutria spp.

Impacts of invasive alien plant species are threatening biodiversity worldwide and thus it is important to assess their effects on particular groups of organisms. However, such impacts were studied mostly in case of plant or invertebrate communities and our understanding the response of vertebrate species to plant invasions remains incomplete. To improve our knowledge in this respect, we studied bird communities in riparian vegetation along the rivers with different levels of Reynoutria spp. invasion in the Czech Republic. These findings will be interesting for basic ecology enhancing our knowledge of consequences of plant invasions, as well as for conservation practice. We surveyed understory bird species in 26 vegetation blocks along parts of three rivers running from the Beskydy Mountains in spring 2011. We used principal component analysis to assess vegetation structure of particular blocks and the first axis ordinated the blocks according to the degree of invasion by Reynoutria spp. Using generalized linear mixed-effects models we found that counts of Motacilla cinerea, Cinclus cinclus and Sylvia borin, as well as the total bird species richness, significantly decreased with increasing degree of Reynoutria spp. invasion, while Acrocephalus palustris showed the opposite pattern. These results suggest that Reynoutria spp. impacts negatively on the species strictly bond with river banks and habitats specialists, whereas habitat generalist species like Sylvia atricapilla were not affected. Preference of Acrocephalus palustris for Reynoutria spp. corroborates affinity of this species to large invasive herbs observed also in other studies. Our study showed that Reynoutria spp. invasion can reduce species richness of understory birds in riparian communities. Although the distribution of this plant species is still quite limited in central Europe, our results suggest that its more widespread occurrence could potentially threat some river bank bird species. Therefore, we urge for development of management actions that will act counter the Reynoutria spp. invasion.     

Diversity of Xenorhabdus and Photorhabdus spp. and Their Symbiotic Entomopathogenic Nematodes from Thailand

Xenorhabdus and Photorhabdus spp. are bacterial symbionts of entomopathogenic nematodes (EPNs). In this study, we isolated and characterized Xenorhabdus and Photorhabdus spp. from across Thailand together with their associated nematode symbionts, and characterized their phylogenetic diversity. EPNs were isolated from soil samples using a Galleria-baiting technique. Bacteria from EPNs were cultured and genotyped based on recA sequence. The nematodes were identified based on sequences of 28S rDNA and internal transcribed spacer regions. A total of 795 soil samples were collected from 159 sites in 13 provinces across Thailand. A total of 126 EPNs isolated from samples taken from 10 provinces were positive for Xenorhabdus (n = 69) or Photorhabdus spp. (n = 57). Phylogenetic analysis separated the 69 Xenorhabdus isolates into 4 groups. Groups 1, 2 and 3 consisting of 52, 13 and 1 isolates related to X. stockiae, and group 4 consisting of 3 isolates related to X. miraniensis. The EPN host for isolates related to X. stockiae was S. websteri, and for X. miraniensis was S. khoisanae. The Photorhabdus species were identified as P. luminescens (n = 56) and P. asymbiotica (n = 1). Phylogenenic analysis divided P. luminescens into five groups. Groups 1 and 2 consisted of 45 and 8 isolates defined as subspecies hainanensis and akhurstii, respectively. One isolate was related to hainanensis and akhurstii, two isolates were related to laumondii, and one isolate was the pathogenic species P. asymbiotica subsp. australis. H. indica was the major EPN host for Photorhabdus. This study reveals the genetic diversity of Xenorhabdus and Photorhabdus spp. and describes new associations between EPNs and their bacterial symbionts in Thailand.     

Specific Immune Response of Mares and their Newborn Foals to Actinobacillus spp. Present in the Oral Cavity

Oral swab samples, serum and colostrum was taken from 15 mares and 14 of their foals, within 24 h of birth. The presence of antibody against Actinobacillus spp. isolated from the oral cavity was investigated using agar gel immunodiffusion. Antibodies against 48 out of the 77 Actinobacillus isolates from all horses in the study were present in the respective sera of 13 mares and 9 foals. In 11 mother-foal pairs, the antibody content of the foal serum was similar to that of the mare, and in 9 cases this was reflected in the antibody content of colostrum from the mare. The results indicate that an immune response to Actinobacillus spp. colonising the oral cavity is present in many adult horses and that this immune response can be transferred from mother to foal via colostrum.     

Diversity and antibiotic resistance of Acinetobacter spp. in water from the source to the tap

Acinetobacter spp. are ubiquitous bacteria in the environment. Acinetobacter spp. isolated from a municipal drinking water treatment plant and from connected tap water were identified to the species level on the basis of rpoB gene partial sequence analysis. Intraspecies variation was assessed based on the analysis of partial sequences of housekeeping genes (rpoB, gyrB, and recA). Antibiotic resistance was characterized using the disk diffusion method and isolates were classified as wild or non-wild type (non-WT), according to the observed phenotype. The strains of Acinetobacter spp. were related to 11 different validly published species, although three groups of isolates, presenting low rpoB sequence similarities with previously described species, may represent new species. Most of the isolates were related to the species A. johnsonii and A. lwoffii. These two groups, as well as others related to the species A. parvus and A. tjernbergiae, were detected in the water treatment plant and in tap water. Other strains, related to the species A. pittii and A. beijerinckii, were isolated only from tap water. Most of the isolates (80 %) demonstrated wild type (WT) to all of the 12 antibiotics tested. Non-WT for tetracycline, meropenem, and ceftazidime, among others, were observed in water treatment plant or in tap water samples. Although, in general, this study suggests a low prevalence of acquired antibiotic resistance in water Acinetobacter spp., the potential of some species to acquire and disseminate resistance via drinking water is suggested.     

The Generation of Successive Unmarked Mutations and Chromosomal Insertion of Heterologous Genes in Actinobacillus pleuropneumoniae Using Natural Transformation

We have developed a simple method of generating scarless, unmarked mutations in Actinobacillus pleuropneumoniae by exploiting the ability of this bacterium to undergo natural transformation, and with no need to introduce plasmids encoding recombinases or resolvases. This method involves two successive rounds of natural transformation using linear DNA: the first introduces a cassette carrying cat (which allows selection by chloramphenicol) and sacB (which allows counter-selection using sucrose) flanked by sequences to either side of the target gene; the second transformation utilises the flanking sequences ligated directly to each other in order to remove the cat-sacB cassette. In order to ensure efficient uptake of the target DNA during transformation, A. pleuropneumoniae uptake sequences are added into the constructs used in both rounds of transformation. This method can be used to generate multiple successive deletions and can also be used to introduce targeted point mutations or insertions of heterologous genes into the A. pleuropneumoniae chromosome for development of live attenuated vaccine strains. So far, we have applied this method to highly transformable isolates of serovars 8 (MIDG2331), which is the most prevalent in the UK, and 15 (HS143). By screening clinical isolates of other serovars, it should be possible to identify other amenable strains.     

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.     

Inhibition of Adherence of Actinobacillus pleuropneumoniae to Porcine Respiratory Tract Cells by Monoclonal Antibodies Directed Against LPS and Partial Characterization of the LPS Receptors

Actinobacillus pleuropneumoniae is the causative agent of porcine fibrinohemorrhagic necrotizing pleuropneumonia. We have previously identified the lipopolysaccharides (LPS) as the major adhesin of A. pleuropneumoniae involved in adherence to porcine respiratory tract cells. In the present study, adherence of A. pleuropneumoniae to porcine tracheal frozen sections was inhibited by homologous monovalent Fab fragments produced from monoclonal antibodies 5.1 G8F10 and 102-G02 directed, respectively, against the A. pleuropneumoniae serotype 1 or serotype 2 O-antigens. These results confirm the important role played by LPS in adherence of A. pleuropneumoniae and suggest that these adhesins might represent good vaccine candidates. We also investigated the presence of A. pleuropneumoniae receptors in tracheal cell preparations from piglets of four different breeds. Using Far-Western binding assays, we identified proteins recognized by whole cells of A. pleuropneumoniae reference strains for serotype 1 and 2, and local isolates belonging to the same serotypes, and also recognized by extracted LPS from both reference strains. We confirmed the proteinaceous nature of these LPS-binding molecules by their staining with Coomassie brilliant blue, sensitivity to proteinase K digestion, resistance to sodium m-periodate oxidation, and their inability to stain with glycoprotein-specific reagents. Four low-molecular-mass bands (14–17 kDa) seemed to correspond to histones. We also identified proteins at Mr 38,500 that could represent putative receptors for A. pleuropneumoniae LPS in swine respiratory tract cells. Received: 16 April 1999 / Accepted: 1 July 1999