Characterization of colonization-deficient mutants of Actinobacillus suis.

Actinobacillus suis is an important opportunistic pathogen of swine that can cause disease in pigs of all ages, especially in high-health status herds. Although A. suis shares many virulence factors in common with Actinobacillus pleuropneumoniae and can cause a haemorrhagic pleuropneumonia similar to that caused by A. pleuropneumoniae, A. suis most often causes septicaemia and diseases such as arthritis and meningitis that are sequelae to septicaemia. In a recent signature-tagged transposon mutagenesis study, 30 colonization-essential genes of A. suis were identified. In the current study, the attachment and invasion patterns of strains harboring Tn10 insertions in ompA, pfhaB1, lcbB, and cpxR were evaluated using porcine palatine tonsil organ cultures, the swine kidney epithelial cell line, SK6, and a porcine brain microvascular endothelial cell line, PBMEC/C1-2. All of these mutants attached in lower numbers than wild type to the tonsillar explants and to the SK6 cells. The ompA mutant attached in significantly lower numbers than wild type to the porcine tonsil cells (P=0.02) and to PBMEC (P=0.0008) at 60 min time point. As well, the ompA mutant showed significantly greater sensitivity than wild type to chemical stressors and to swine serum. Using fluorescent microscopy, a GST-OmpA fusion protein could be demonstrated to interact with the crypt epithelial cells of porcine palatine tonsil.

A search for virulence genes of Haemophilus parasuis using differential display RT-PCR.

Although Haemophilus parasuis is an important bacterial pathogen of swine, little is known about its pathogenesis or why some strains seem to be more virulent than others. Therefore, we used differential display reverse transcription-polymerase chain reaction (DDRT-PCR) to search for virulence-associated genes in a pathogenic serotype 5 strain, H. parasuis 1185. Gene expression was evaluated following growth in conditions chosen to begin to approximate those found in the upper respiratory tract and those encountered by the organism during acute infection. Seven different differentially expressed gene fragments were identified in cells grown at 40 degrees C in both the presence and absence of swine serum. Based on the deduced amino acid sequences, the most strongly up-regulated genes were homologs of fadD (a fatty acyl-CoA synthetase), apaH (diadenosine tetraphosphatase), pstI (enzyme I of the phosphoenolpyruvate-protein phosphotransferase system), and cysK (cysteine synthetase). Homologs of Std (Na(+)- and Cl(-)-dependent ion transporter), HSPG (a mammalian basement membrane-specific heparin sulphate core protein precursor) and PntB (pyridine nucleotide transhydrogenase) were also up-regulated, but to a much lower extent. Sequences homologous to all of the differentially expressed genes were detected in the reference strains of all 15 H. parasuis serotypes. This is the first report of a global search for virulence factors of H. parasuis.

Novel genes affecting urease acivity in Actinobacillus pleuropneumoniae.

Characterization of a series of urease-negative transposon mutations of Actinobacillus pleuropneumoniae revealed that many of the mutants had insertions 2 to 4 kbp upstream of the urease gene cluster. A 5-kbp upstream region of DNA was sequenced and found to contain six open reading frames (ORFs) transcribed in the same orientation as the urease genes. As well, a partial ORF, apuR, 202 bp upstream of these six ORFs, is transcribed in the opposite orientation. The predicted product of this partial ORF shows homology with many members of the LysR family of transcriptional regulators. Five of the ORFs (cbiKLMQO) appear to form an operon encoding a putative nickel and cobalt periplasmic permease system. The cbiM and cbiQ genes encode proteins that have sequence similarity with known cobalt transport membrane proteins, and the cbiO gene encodes a cobalt transport ATP-binding protein homologue. The product of the cbiK gene is predicted to be the periplasmic-binding-protein component of the system, though it does not show any sequence similarity with CbiN, the cobalt-binding periplasmic protein. Escherichia coli clones containing this putative transport operon together with the urease genes of A. pleuropneumoniae were urease positive when grown in unsupplemented Luria-Bertani broth, whereas a clone containing only the minimal urease gene cluster required the addition of high concentrations of NiCl(2) for full urease activity. This result supports the hypothesis that nickel is a substrate for this permease system. The sixth ORF, utp, appears to encode an integral membrane protein which has significant sequence identity with mammalian urea transport proteins, though its function in A. pleuropneumoniae remains to be determined.

The first description of a (1--> 6)-beta-D-glucan in prokaryotes: (1 --> 6)-beta-D-glucan is a common component of actinobacillus suis and is the basis for a serotyping system.

The chemical and antigenic properties of the cell-surface lipopolysaccharides (LPSs) and capsular polysaccharides (CPSs) of seven representative strains of Actinobacillus suis from healthy and diseased pigs were investigated. Four strains produced a linear (1 --> 6)-beta-D-glucan homopolymer, beta-D-Glcp-(1-[ --> 6)-beta-D-Glcp-(1-]n -->, as a LPS-O-chain (O1) and as a CPS (K1). Polyclonal antisera prepared against a (1 --> 6)-beta-D-glucan-containing strain showed a positive reaction against both LPSs and CPSs derived from the above strains (designated serotype O1/K1). One strain carried the (1 --> 6)-beta-D-glucan solely as a LPS-O-chain (serotype O1) and two strains did not express the (1 --> 6)-beta-D-glucan, but, instead, produced a different O-chain (designated serotype 02); these three strains expressed their own characteristic CPSs. (1 --> 6)-beta-D-Glucan structures are common cell wall components of yeast, fungi and lichens, but, to our knowledge, this is the first time a (1 --> 6)-beta-D-glucan has been described in a prokaryotic organism. Conformational and nuclear magnetic resonance analyses showed that the beta-D-Glcp-(1 --> 6)-beta-D-Glcp linkage was flexible and two distinct glycosidic conformers are described. Cross-reactive antibodies to the A. suis (1 --> 6)-beta-D-glucan could be detected in sera from a variety of species and in sera from specific pathogen free pigs. This cross-reactivity may arise from immuno-stimulation of organisms present in the surrounding environment that contain (1 --> 6)-beta-D-glucan, which may also explain the high incidence of false positive results in previous serological tests for A. suis. In addition, these (1 --> 6)-beta-D-glucan background antibodies may be protective against A. suis infection. The characterization herein of (1 --> 6)-beta-D-glucan is the foundation for the development of a serotyping system for A. suis.

Prevalence of O1/K1- and O2/K3-reactive Actinobacillus suis in healthy and diseased swine.

A cell surface antigen-typing system was devised for the swine pathogen Actinobacillus suis and used to examine the prevalence of different lipopolysaccharide (O) types in healthy and diseased pigs. The strains examined in this study were isolated from a variety of locations in Canada and from Kansas. Lipopolysaccharide preparations of 151 isolates of A. suis were characterized by immunoblotting using polyclonal antisera generated to strains SO4 (O1/K1), H89-1173 (O2/K3), and VSB 3714, a rough strain. Approximately 54% (62 of 114) of A. suis isolates from diseased pigs, all (11 of 11) isolates from healthy pigs, and all (4 of 4) reference strains reacted with O1/K1 antiserum. More than 80% (18 of 22) of A. suis strains used for bacterin production and approximately 41% (47 of 114) of isolates from diseased pigs bound O2/K3 antiserum. One isolate appeared to be rough, and five were untypeable. O1/K1- and O2/K3-reactive strains were equally prevalent in Kansas, whereas O2/K3-reactive strains were more common in Québec and western Canada and O1/K1 strains were most common in Ontario. The fact that virtually all of the strains submitted for bacterin production were O2/K3-reactive strains is consistent with the notion that these strains may be more virulent than O1/K1 strains; alternatively, this may reflect geographic or other biases. In addition, we observed cross-reactivity between A. suis cell surface antigens and swine antisera to several other important pathogens. This finding may explain why previous attempts to develop a simple serodiagnostic test for A. suis have been unsuccessful.

Urease activity may contribute to the ability of Actinobacillus pleuropneumoniae to establish infection.

The contribution of urease activity to the pathogenesis of Actinobacillus pleuropneumoniae was investigated using 2 different urease-negative transposon mutants of the virulent serotype 1 strain, CM5 Nalr. One mutant, cbiK::Tn10, is deficient in the uptake of nickel, a cofactor required for urease activity. The other mutant, ureG::Tn10, is unable to produce active urease due to mutation of the urease accessory gene, ureG. In aerosol challenge experiments, pigs developed acute pleuropneumonia following exposure to high doses (10(6) cfu/mL) of the parental strain, CM5 Nalr, and to the cbiK::Tn10 mutant. When low dose (10(3) cfu/mL) challenges were used, neither urease-negative mutant was able to establish infection, whereas the parental strain was able to colonize and cause lesions consistent with acute pleuropneumonia in 8 of the 20 pigs challenged. These findings suggest that urease activity may be needed for A. pleuropneumoniae to establish infection in the respiratory tract of pigs.

Comparative pathogenicity of different Actinobacillus suis O/K serotypes.

The pathogenicity of Actinobacillus suis serotypes O1/K1 (strain SO4), O1/K2 (strain C84), and O2/K2 (strain H91-0380) was evaluated in specific-pathogen-free (SPF) piglets challenged by intraperitoneal inoculation with approximately 1 x 10(7) colony-forming units per mL. All 3 strains produced peritonitis, but differences were observed in the composite histopathologic scores (P = 0.001) and in their ability to spread (P = 0.008) at 7 h post challenge. The O2/K2 strain caused the most severe peritonitis and disseminated most widely to other tissues. Moderate lesions were seen with the O1/K2 strain while the O1/K1 strain caused mild lesions and remained largely localized to the peritoneum. In an attempt to explain the basis of observed differences, the serum sensitivity of 9 A. suis strains with different O and K types was assessed. Regardless of the O/K type, all of the isolates tested were serum resistant. Moreover, most A. suis isolates grew as well or better in complement-replete sera as they did in complement-depleted sera. These observations indicate that although 02 and K2 strains had a greater propensity to cause a disseminating septic inflammatory response in pigs, they were no more resistant to complement-mediated killing than O1 strains.

Agents of the "suis-ide diseases" of swine: Actinobacillus suis, Haemophilus parasuis, and Streptococcus suis.

In recent years, Actinobacillus suis, Haemophilus parasuis, and Streptococcus suis have emerged as important pathogens of swine, particularly in high health status herds. Their association with a wide range of serious clinical conditions and has given rise to the moniker "suis-ide diseases." These organisms are early colonizers and, for that reason, are difficult to control by management procedures such as segregated early weaning. Vaccination, serodiagnostic testing, and even serotyping are complicated by the presence of multiple serotypes, cross-reactive antigens, and the absence of clear markers for virulence. In this review, we discuss our current understanding of the pathogenesis, epidemiology, and management of the causative agents of the "suis-ide diseases" of swine.

Anti-haemolysin IgG1 to IgG2 ratios correlate with haemolysin neutralization titres and lung lesion scores in Actinobacillus pleuropneumoniae infected pigs.

Currently available porcine Actinobacillus pleuropneumoniae bacterins afford only minimal protection by decreasing mortality but not morbidity. To evaluate a possible role of IgG subclasses in protection, IgG1 and IgG2 responses to A. pleuropneumoniae haemolysin (HLY) were examined in piglets exposed to a low dose (10(5) c.f.u. ml-1) of A. pleuropneumoniae CM5 (LD) given by aerosol (which affords complete protection) or bacterin-vaccinated piglets (no protection). Only the LD group developed HLY neutralizing antibody. These animals produced both IgG1 and IgG2-associated antibody in response to HLY, and there was a positive correlation (r = 0.6) between IgG1 anti-HLY antibody and neutralizing titres. Anti-HLY IgG1 antibody was negatively correlated with pneumonic scores at necropsy (r = -0.67, p < or = 0.005). These results suggest that immunization procedures that bias anti-HLY antibody to IgG1 may be more efficacious than those inducing IgG2.

Genetic and biochemical analyses of Actinobacillus pleuropneumoniae urease.

The urease gene cluster from the virulent Actinobacillus pleuropneumoniae serotype 1 strain CM5 was cloned and sequenced. The urease activity was associated with a 6.3-kbp region which contains eight long open reading frames (ORFs). The structural genes, ureABC, are separated from the accessory genes, ureEFGD, by a 615-bp ORF of unknown function, ureX. Homologies were found with the structural and accessory urease gene products of Haemophilus influenzae and, to a lesser extent, with those of other organisms. The urease enzyme subunits had predicted molecular masses of 61.0, 11.3, and 11.0 kDa, and the size of the holoenzyme was estimated to be 337 +/- 13 kDa by gel filtration chromatography. Urease activity was maximal but unstable at 65 degrees C. In cell lysates, the A. pleuropneumoniae urease was stable over a broad pH range (5.0 to 10.6) and the optimal pH for activity was 7.7. The Km was 1.5 +/- 0.1 mM urea when it was assayed at pH 7.7. The low Km suggests that this enzyme would be active in the respiratory tract environment, where urea levels should be similar to those normally found in pig serum (2 to 7 mM).

Expression and phylogenetic relationships of a novel lacZ homologue from Actinobacillus pleuropneumoniae.

To learn more about the genetics and physiology of the important swine pathogen, Actinobacillus pleuropneumoniae, we cloned the lacZ gene by complementation of an Escherichia coli delta lac mutant. The A. pleuropneumoniae lacZ gene has an open reading frame of 3015 bp which could encode a protein with a predicted molecular mass of 117022. The deduced protein shares 26.8-34.8% identity with beta-galactosidases from both Gram-positive and Gram-negative bacteria. Sequences with homology to seven regions commonly found in beta-galactosidases are present and amino acids corresponding to active site residues Tyr-503 and Glu-537 in E. coli LacZ are also conserved; however, there is a leucine in the place of Gly-794, a residue which has been implicated in substrate recognition. The sequences flanking the A. pleuropneumoniae lacZ gene do not share homology with known transport or regulatory genes nor do they share homology with cAMP receptor protein (CRP) or LacI binding sites. Low levels of beta-galactosidase activity could be detected when the protein was expressed from a multicopy plasmid in E. coli delta lac and when it was measured in A. pleuropneumoniae. The level of activity was not markedly reduced in the presence of glucose. Although the A. pleuropneumoniae LacZ shares some features with other beta-galactosidases, its constitutive expression and an unusual active site residue suggest that it may have a unique function.

Actinobacillus suis strains isolated from healthy and diseased swine are clonal and carry apxICABDvar. suis and apxIICAvar. suis toxin genes.

Actinobacillus suis isolates recovered from both healthy and diseased pigs were characterized by biochemical testing, serotyping, restriction endonuclease fingerprinting, and apx toxin gene typing. The clinical isolates analyzed were collected over a 10-year period from approximately 40 different locations in southwestern Ontario, Canada. Little variation in the biochemical profiles of these isolates was seen, and all isolates reacted strongly with rabbit antisera prepared against one of the strains. Similarly, by using BamHI and BglII for restriction endonuclease fingerprinting (REF) analysis, all isolates were found to belong to a single REF group. Minor variations could be detected, especially in the BglII fingerprints, but overall the patterns were remarkably similar. Sequences that could be amplified by PCR with primers to the apxICA and apxIICA genes of Actinobacillus pleuropneumoniae were detected in all strains. Although no amplification was obtained with primers to the A. pleuropneumoniae apxIBD genes, sequences with homology to apxIBD were detected by hybridization. There was no evidence of apxIII homologs. Taken together, these data suggest that A. suis isolates are genotypically and phenotypically very similar, regardless of their source, and that they contain genes similar to, but not identical to, the apxICABD and apxIICA genes of A. pleuropneumoniae.

Antibody- and cell-mediated immune responses of Actinobacillus pleuropneumoniae-infected and bacterin-vaccinated pigs.

Current porcine pleuropneumonia bacterins afford only partial protection by decreasing mortality but not morbidity. In order to better understand the type(s) of immune response associated with protection, antibody- and cell-mediated immune responses (CMIR) were compared for piglets before and after administration of a commercial bacterin, which confers partial protection, or a low-dose (10(5) CFU/ml) aerosol challenge with Actinobacillus pleuropneumoniae CM5 (LD), which induces complete protection. Control groups received phosphate-buffered saline or adjuvant. Serum antibody response, antibody avidity, delayed-type hypersensitivity (DTH), and lymphocyte blastogenic responses were measured and compared among treatment groups to the lipopolysaccharide (LPS), capsular polysaccharide (CPS), hemolysin (HLY), and outer membrane proteins (OMP) of A. pleuropneumoniae. Peripheral blood lymphocytes and sera were collected prior to and following primary and secondary immunization-infection and high-dose A. pleuropneumoniae CM5 (10(7) CFU/ml) aerosol challenge. Serum antibody and DTH, particularly that to HLY, differed significantly between treatment groups, and increases were associated with protection. LD-infected piglets had higher antibody responses (P < or = 0.01) and antibody avidity (P < or = 0.10) than bacterin-vaccinated and control groups. Anti-HLY antibodies were consistently associated with protection, whereas anti-LPS and anti-CPS antibodies were not. LD-infected animals had higher DTH responses, particularly to HLY, than bacterin-vaccinated pigs (P < or = 0.03). The LD-infected group maintained consistent blastogenic responses to HLY, LPS, CPS, and OMP over the course of infection, unlike the bacterin-vaccinated and control animals. These data suggest that the immune responses induced by a commercial bacterin are very different from those induced by LD aerosol infection and that current bacterins may be modified, for instance, by addition of HLY, so as to stimulate responses which better reflect those induced by LD infection.

Regulation of gene expression by the HlyX protein of Actinobacillus pleuropneumoniae.

The hlyX gene of the swine pathogen Actinobacillus pleuropneumoniae and the fnr gene of Escherichia coli encode very similar proteins. The hlyX gene is able to complement delta fnr mutations and will permit the growth of E. coli fnr strains in nitrate minimal salts medium under anoxic conditions. In addition, the hlyX gene product appears to induce the expression of a latent haemolytic activity as evidenced by the presence of a strong zone of haemolysis around E. coli (hlyX+) colonies grown on bovine or ovine blood. In this study, the ability of the hlyX gene product to induce haemolytic activity and regulate expression of frdA and its own gene was examined in the presence of various carbon sources and in the presence and absence of iron; fnr was included for comparison. The HlyX protein was able to induce the synthesis of the latent E. coli haemolytic activity only under anoxic conditions. Haemolytic activity was highest during the late exponential phase and then levelled off in the stationary phase. The hlyX gene product was able to activate the expression of a phi (frdA'-lacZ) in E. coli JRG1787 (delta fnr); however, the level of expression depended on carbon source, growth phase and copy number. Like fnr, the hlyX gene product appeared to affect its own synthesis but the nature and extent of regulation depended not only on the presence of oxygen but also on growth conditions.

Preliminary characterization of the structural proteins of the coronaviruses, sialodacryoadenitis virus and Parker's rat coronavirus.

A procedure was developed for the partial purification of the rat coronaviruses, sialodacryoadenitis virus (SDAV) and Parker's rat coronavirus (PRC). The SDAV and PRC were replicated in L-2 cell monolayer cultures, precipitated with ammonium sulphate, and further concentrated using sucrose density gradient centrifugation. The major SDAV and PRC proteins were identified by immunoblotting and compared with those of the JHM strain of mouse hepatitis virus (MHV-JHM). Monoclonal antibodies (MAb) against the M protein of JHM recognized proteins interpreted to be slightly smaller in immunoblots of SDAV and PRC (22.8 vs 23K for JHM). Similarly, a monoclonal antibody against the JHM N protein reacted with proteins of 53K in SDAV and PRC (vs 56 K for JHM). Polyclonal antisera to all three viruses also cross-reacted with the M and N proteins. Some cross-reactivity amongst the S proteins was observed. Based on these data, the structural proteins of the rat coronaviruses, SDAV and PRC are closely related to those of MHV-JHM.

Cloning and characterization of btr, a Bordetella pertussis gene encoding an FNR-like transcriptional regulator.

To determine whether hemolytic factors other than the bifunctional hemolysin-adenylate cyclase toxin (cyclolysin) are expressed by Bordetella pertussis, a gene library was constructed from a virulent strain of B. pertussis, BP504, transformed into nonhemolytic Escherichia coli, and screened on blood agar plates. A strongly hemolytic colony which contained the plasmid pHLY1A was isolated. Nucleotide sequencing of pHLY1A revealed an open reading frame that could encode a 27-kDa protein. No similarity was detected between the deduced amino acid sequence of this open reading frame and those of any known bacterial cytolysins. However, significant homology was detected with FNR of E. coli and several other transcriptional regulators including HylX from Actinobacillus pleuropneumoniae, which can also confer a hemolytic phenotype on E. coli. An fnr mutant of E. coli, JRG1728, could be complemented by pHLY1A. Thus, the B. pertussis transcriptional regulator-like gene and the protein which it encoded were named btr and BTR, respectively. A BTR-deficient B. pertussis strain, BJB1, was constructed. The btr::kan mutation had no effect on the expression of hemolytic activity or on phase variation. Northern (RNA) blotting revealed that btr expression was not regulated by the BvgAS two-component sensor-regulator. On the basis of sequence similarity to FNR-like transcriptional regulators and the ability to complement an anaerobically deficient E. coli strain (JRG1728) in growing anaerobically, BTR may regulate B. pertussis gene expression in response to changes in oxygen levels or to changes in the redox potential of the bacterial environment. Its role in virulence remains to be determined.

An investigation of enzootic Glasser's disease in a specific-pathogen-free grower-finisher facility using restriction endonuclease analysis.

Enzootic Glassers's disease was investigated to study the epidemiology of the disease strains on a farm where it presented a problem. Restriction endonuclease fingerprinting (REF) analysis technique was used, as all strains of Haemophilus parasuis are biochemically similar and many strains are biochemically untypable. After young weaned pigs were moved from farm A to farm B, Glasser's disease routinely occurred despite the use of antibiotics and a commercial bacterin. Isolates were taken from the nasal passages and from carcasses of clinically affected cases and subjected to REF analysis. Haemophilus parasuis was not isolated from any of the pigs on farm A, but it was isolated from 7/10 and 5/10 nasal swabs taken from farm B. Two H. parasuis strains isolated from clinical cases of Glasser's disease from farm B had an identical REF pattern, but were different from the nasal swabs and the H. parasuis strain contained in the bacterin. The subsequent use of a custom autogenous bacterin made from a clinical isolate of H. parasuis reduced the mortality rate on farm B. This investigation indicates that nasal isolates of H. parasuis are different than those causing clinical disease, and not all bacterin strains are cross protective for other strains.

Molecular analysis of isolates of Streptococcus suis capsular type 2 by restriction-endonuclease-digested DNA separated on SDS-PAGE and by hybridization with an rDNA probe.

This study was undertaken to assess the discriminatory value of restriction endonuclease (RE) digestion patterns of Streptococcus suis chromosomal DNA using polyacrylamide gel electrophoresis (SDS-PAGE) and DNA-rDNA hybridization. For the RE digestion patterns, DNAs were digested separately with the enzymes BamHI and BglII and the resultant fragments were separated by SDS-PAGE. An Escherichia coli rDNA probe derived from pKK3535 was used for the hybridization. Twenty-three S. suis capsular type 2 isolates recovered from diseased and clinically healthy pigs, from a human case, and from a cow were compared in this study. The majority of isolates associated with septicaemia belonged to one restriction endonuclease analysis (REA) profile group. Isolates associated with pneumonia belonged either to the REA profile group of isolates associated with septicaemia or to a second REA profile group. The REA profiles of isolates from clinically healthy animals were more heterogeneous. The REA profile of the type 2 reference strain, S735, which was originally isolated from a pig, was very different from those of the porcine and bovine isolates but similar to the profile of the human isolate. The profiles obtained after rDNA hybridization were more homogeneous. Although different patterns were detected in the 23 isolates, there was no correlation between the source of the isolate and the patterns observed with this technique.

Purification of HlyX, a potential regulator of haemolysin synthesis, and properties of HlyX:FNR hybrids.

The hlyX gene of the swine pathogen Actinobacillus pleuropneumoniae is homologous to FNR, an anaerobic transcriptional regulator of Escherichia coli. It endows a haemolytic phenotype upon E. coli, and will complement the anaerobic respiratory deficiencies of fnr mutants of E. coli. The coding region of the hlyX gene was expressed in E. coli and the HlyX protein was purified by using an assay based on its immunological cross-reactivity with anti-FNR antibodies. The HlyX protein had the predicted N-terminal sequence, and resembled the isolated FNR protein in size (Mr 29,000) and monomeric organization. It has no detectable haemolysin activity per se, and is therefore presumed to confer a haemolytic phenotype by activating a latent haemolysin gene in E. coli. Studies with gene fusions showed that HlyX, like FNR, can function as an anaerobic activator and repressor of FNR-regulated genes in vivo. Plasmids that express hybrid HlyX:FNR proteins in which the 189/190-residue N-terminal segments and the remaining 50/60-residue C-terminal segments are exchanged, retained their FNR-specific functions but failed to confer a haemolytic phenotype. This suggests that the specificity for activating the haemolytic response requires the participation of unique features in both the N- and C-terminal segments of HlyX.

Analysis of Actinobacillus pleuropneumoniae and related organisms by DNA-DNA hybridization and restriction endonuclease fingerprinting.

The objective of this study was to determine the degree of genetic relatedness of Actinobacillus pleuropneumoniae to selected members of the family Pasteurellaceae, with particular emphasis on species commonly associated with swine. Free-solution DNA-DNA hybridization studies revealed that representative strains of all 12 serotypes of A. pleuropneumoniae formed a homogeneous group, sharing 74 to 90% sequence homology with A. pleuropneumoniae serotype 1. All serotypes of A. pleuropneumoniae tested demonstrated a high degree of genetic relatedness (66 to 79%) to the type species of the genus Actinobacillus, A. lignieresii. Little homology (less than 20%) was detected between A. pleuropneumoniae strains and selected Haemophilus spp. and Pasteurella spp. Since free-solution hybridization methods are technically demanding and require large amounts of highly purified DNA, restriction endonuclease fingerprinting (REF) was examined to determine whether it could be a useful taxonomic tool for classification of members of the family Pasteurellaceae. REF profiles were compared, and the degree of similarity between organisms was quantitated by calculating Jaccard similarity coefficients. There was a significant positive relationship between the REF Jaccard coefficients and the DNA homology values determined from free-solution hybridization experiments.