Investigation of putative invasion determinants of Actinobacillus species using comparative genomics.

Actinobacillus spp. are Gram-negative bacteria associated with mucosal membranes. While some are commensals, others can cause important human and animal diseases. A. pleuropneumoniae causes severe fibrinous hemorrhagic pneumonia in swine but not systemic disease whereas other species invade resulting in septicemia and death. To understand the invasive phenotype of Actinobacillus spp., complete genomes of eight isolates were obtained and pseudogenomes of five isolates were assembled and annotated. Phylogenetically, A. suis isolates clustered by surface antigen type and were more closely related to the invasive A. ureae, A. equuli equuli, and A. capsulatus than to the other swine pathogen, A. pleuropneumoniae. Using the LS-BSR pipeline, 251 putative virulence genes associated with serum resistance and invasion were detected. To our knowledge, this is the first genome-wide study of the genus Actinobacillus and should contribute to a better understanding of host tropism and mechanisms of invasion of pathogenic Actinobacillus and related genera.

A genomic perspective on the potential of Actinobacillus succinogenes for industrial succinate production.

BACKGROUND: Succinate is produced petrochemically from maleic anhydride to satisfy a small specialty chemical market. If succinate could be produced fermentatively at a price competitive with that of maleic anhydride, though, it could replace maleic anhydride as the precursor of many bulk chemicals, transforming a multi-billion dollar petrochemical market into one based on renewable resources. Actinobacillus succinogenes naturally converts sugars and CO2 into high concentrations of succinic acid as part of a mixed-acid fermentation. Efforts are ongoing to maximize carbon flux to succinate to achieve an industrial process. RESULTS: Described here is the 2.3 Mb A. succinogenes genome sequence with emphasis on A. succinogenes's potential for genetic engineering, its metabolic attributes and capabilities, and its lack of pathogenicity. The genome sequence contains 1,690 DNA uptake signal sequence repeats and a nearly complete set of natural competence proteins, suggesting that A. succinogenes is capable of natural transformation. A. succinogenes lacks a complete tricarboxylic acid cycle as well as a glyoxylate pathway, and it appears to be able to transport and degrade about twenty different carbohydrates. The genomes of A. succinogenes and its closest known relative, Mannheimia succiniciproducens, were compared for the presence of known Pasteurellaceae virulence factors. Both species appear to lack the virulence traits of toxin production, sialic acid and choline incorporation into lipopolysaccharide, and utilization of hemoglobin and transferrin as iron sources. Perspectives are also given on the conservation of A. succinogenes genomic features in other sequenced Pasteurellaceae. CONCLUSIONS: Both A. succinogenes and M. succiniciproducens genome sequences lack many of the virulence genes used by their pathogenic Pasteurellaceae relatives. The lack of pathogenicity of these two succinogens is an exciting prospect, because comparisons with pathogenic Pasteurellaceae could lead to a better understanding of Pasteurellaceae virulence. The fact that the A. succinogenes genome encodes uptake and degradation pathways for a variety of carbohydrates reflects the variety of carbohydrate substrates available in the rumen, A. succinogenes's natural habitat. It also suggests that many different carbon sources can be used as feedstock for succinate production by A. succinogenes.

Granulomatous lymphadenitis and pneumonia associated with Actinobacillus porcitonsillarum in a slaughter pig.

Multiple coalescing granulomatous foci were detected in the pulmonary hilar and mediastinal lymph nodes and lung of a slaughtered pig aged 6 months. Haemolytic, Gram-negative bacilli were isolated from the lymph nodes. The isolate (strain TO17214) strongly cross-reacted with sera against Actinobacillus pleuropneumoniae serotype 12 in slide agglutination tests. Comparative 16S rDNA gene sequencing analysis identified strain TO17214 as Actinobacillus porcitonsillarum. Histologically, extensive inflammation took the form of large granulomas consisting of epithelioid cells and multinucleated giant cells in the lymph nodes and lung, and Gram-negative bacilli were discernible in the centres of the lesions. Immunohistochemically, the organisms cross-reacted with polyclonal antibodies against A. pleuropneumoniae serotypes 12 and 2. The results indicated that A. porcitonsillarum, previously considered non-pathogenic, can induce multifocal granulomatous lymphadenitis accompanied by pneumonia in the growing-finishing pig.

Oral pathogens: from dental plaque to cardiac disease.

Oral bacteria exhibit highly specific adherence mechanisms and as a result they colonize and cause disease principally in the oral cavity. Oral pathogens, however, can produce systemic disease and are known causative agents of infective endocarditis. Recent studies have revealed that periodontal disease per se is also a statistically significant risk factor for cardiovascular disease. A link between the two diseases is the secretion and systemic appearance in periodontitis of pro-inflammatory cytokines capable of eliciting effects associated with atherosclerosis and coronary heart disease.

Immunological properties of Actinobacillus pleuropneumoniae hemolysin I.

The 105 kDa hemolysin I protein from Actinobacillus pleuropneumoniae serotype I type strain 4074 (HlyI) was shown by immunoblot analysis to be the predominant immunogenic protein if convalescent field sera or sera from pigs experimentally infected with A. pleuropneumoniae serotype 1 were used. SDS gel- and immunoblot-analysis using total culture, washed cells or culture supernatant showed that HlyI is essentially secreted and is not found attached to the bacteria. Proteins in the 105 kDa range that react strongly with anti-HlyI antibody, are produced by all serotypes and are presumed to be their hemolysins. Sera from pigs experimentally infected with each of the 12 serotypes strongly reacted with HlyI. In addition, some sera from pigs that were confirmed to be negative for A. pleuropneumoniae, also reacted with HlyI as well as with related proteins from Actinobacillus rossii and Actinobacillus suis. These two species produce proteins in the 105 kDa range which cross-react strongly with HlyI. They could be the source of the immunological reactions of the A. pleuropneumoniae-negative sera with HlyI. However, no cross-reactions could be found between HlyI and the Pasteurella haemolytica leukotoxin, the Escherichia coli alpha-hemolysin or related proteins from various hemolytic E. coli strains isolated from pigs. The immunological cross-reactions of HlyI with related proteins from A. rossii, A. suis and possibly from other bacterial species may create uncertainty in interpretation if HlyI is used as the antigen in serodiagnosis of A. pleuropneumoniae.

Grouping of Actinobacillus pleuropneumoniae strains of serotypes 1 through 12 on the basis of their virulence in mice.

Variations in virulence among strains of different serotypes of Actinobacillus pleuropneumoniae were detected on intraperitoneal or intranasal inoculation in mice. In general, strains of serotypes 1, 5, 9, 10 and 11 were found to be highly virulent and those of serotypes 2, 3, 4, 6, 7, 8 and 12 to be less virulent. However, a few strains of serotype 5 caused low mortality in mice while some strains of serotype 3 and 7 were found to be highly virulent. Highly virulent strains of A. pleuropneumoniae were invasive and appeared in the blood within 3 to 6 h of intranasal inoculation. The type specific antigen as detected by the coagglutination test was distributed in lungs, liver, heart and spleen after intraperitoneal inoculation whereas it was mostly concentrated in the lungs after intranasal inoculation. Lowest concentration of boiled whole-cell suspension of A. pleuropneumoniae showing limulus amebocyte lysate activity was variable and independent of the serotype. Mortality caused by boiled whole cell suspension was also variable and serotype independent.

Non-pathogenic Actinobacillus isolates antigenically and biochemically similar to Actinobacillus pleuropneumoniae: a novel species?

Two unusual Actinobacillus isolates were recovered from pigs with no clinical signs, no lesions and no history of swine pleuropneumonia. Two representative strains (9953L55 and 0347) analyzed in this study were initially biochemically and antigenically identified as A. pleuropneumoniae serotypes 1 and 9, respectively, by traditional identification methods. Both strains presented, however, negative results with three A. pleuropneumoniae-specific PCR tests and revealed in particular the absence of the apxIV toxin genes. However, both strains produced and secreted ApxII toxin although they only harbored the toxin genes apxIICA, which is an uncommon feature for any of the known A. pleuropneumoniae serotypes. Upon experimental inoculation of pigs, these strains proved to be totally non-pathogenic. Animals infected with one of the strains produced antibodies that cross-react with A. pleuropneumoniae serotypes 1-9-11-specific LC-LPS ELISA. Phylogenetic analysis based on 16S rRNA gene sequence analysis revealed that these strains form a separate phylogenetic group that is distinct from other Actinobacillus species and is particularly different from A. pleuropneumoniae.

Host cell specific activity of RTX toxins from haemolytic Actinobacillus equuli and Actinobacillus suis.

We assessed and compared host cell specificity of the haemolytic and cytotoxic activity of the RTX toxins from Actinobacillus equuli, an equine pathogen, and Actinobacillus suis, which is pathogenic for pigs. The two bacterial species are closely related, phenotypically as well as phylogenetically, sharing the same 16S rRNA gene sequence. Both species contain specific protein toxins from the family of pore-forming RTX toxins, however, the two species differ in their RTX toxin profiles. Haemolytic A. equuli contains the operon for the Aqx toxin, whereas A. suis harbours genes for ApxI and ApxII. We tested the toxic activity of the corresponding proteins on erythrocytes as well as on lymphocytes isolated from horse and pig blood. The strength of the haemolytic activity for each of the toxins was independent of the origin of erythrocytes. When testing cytotoxic activity, the Aqx protein showed a higher toxic effect for horse lymphocytes than for porcine lymphocytes. On the other hand, ApxI and ApxII showed a strong cytotoxic effect on porcine lymphocytes and a reduced toxicity for horse lymphocytes; the toxicity of ApxII was generally much lower than ApxI. Our results indicate a host species specificity of the toxic activity of RTX toxins Aqx of A. equuli and ApxI and ApxII of A. suis.

Molecular analysis of periodontal pathogens.

Both Bacteroides gingivalis and Actinobacillus actinomycetemcomitans have been implicated in the destruction of periodontal tissues. To understand better the role putative virulence factors from the two bacterial species may play in an infection, the collagenase gene from Bact. gingivalis and the leucotoxin gene from A. actinomycetemcomitans were cloned. As it is intended to generate strains carrying defined mutations in these genes for in vitro and in vivo experiments, extensive restriction mapping and sequence analyses of these clones are being undertaken. Furthermore a conjugation system for Bact. gingivalis and A. actinomycetemcomitans will be established.

Genetic approach to the study of epidemiology and pathogenesis of Actinobacillus actinomycetemcomitans in localized juvenile periodontitis.

Actinobacillus actinomycetemcomitans isolates from periodontal pockets were examined for restriction fragment-length polymorphism using a characterized 4.7-kb DNA probe. A total of 6 patterns of RFLP was found in 133 isolates originating from 12 subjects. No relatedness was found between RFLP types and serotypes. Different periodontal sites within the same subject and different individuals within the same family sometimes showed only one type of A. actinomycetemcomitans RFLP. When members among the same family showed 2 RFLP types, children were always infected with the A. actinomycetemcomitans strains found in at least one of the parents. These findings support the concept of familial spread of A. actinomycetemcomitans. A. actinomycetemcomitans RFLP type B, corresponding to reference strain JP2, seems to be particularly virulent, as indicated from the presence of RFLP type B in 3 subjects who converted from a healthy periodontal state to localized juvenile periodontitis. RFLP type B was not detected in any of the 21 A. actinomycetemcomitans-infected patients with adult periodontitis. The RFLP method seems to be useful in determining the epidemiology and possibly the potential virulence of periodontal strains of A. actinomycetemcomitans.

Molecular approaches to leucotoxin as a virulence component in Actinobacillus actinomycetemcomitans.

A strategy has been developed to examine the hypothesis that leucotoxin is a critical virulence factor of Actinobacillus actinomycetemcomitans in a non-human primate (Macaca fascicularis). Firstly the leucotoxin gene from A. actinomycetemcomitans was cloned and sequenced. This DNA contained a functional leucotoxin gene, as protein extracts of Escherichia coli with the cloned sequences lysed appropriate human cell lines. The protein encoded by lktA shared at least 42% identity with P. haemolytica leucotoxin and with the alpha-haemolysins from E. coli and A. pleuropneumoniae. The lktA gene of A. actinomycetemcomitans was linked to another gene, lktC, which is thought to be related to the LktC proteins from these other bacteria and with which it shared at least 49% amino acid identity. Despite the overall homology to the other leucotoxins/haemolysins, the LktA from A. actinomycetemcomitans has several unique properties including a very basic pI of 9.7, as compared to pIs approx. 6.2 for lktA proteins in other bacteria. Using the cloned genes as probes produced evidence that a TOX- strain contains the leucotoxin gene but fails to transcribe it at high levels. The second avenue of investigation was to develop methods for examining the humoral immune responses in the monkey to bacterial toxins such as lktA. A. actinomycetemcomitans was detected in subgingival plaque samples from approx. 40% of the animals. A. actinomycetemcomitans comprised less than 1% to 9% of the flora. Most A. actinomycetemcomitans isolates were serotype b and each of the monkeys had serum IgG antibody to A. actinomycetemcomitans serotype b (generally considered to be lktA-producing strains). An ELISA was developed to examine the isotype/subclass distribution, level and avidity of serum antibody in the monkey following parenteral immunization with a prototype bacterial exotoxin (tetanus toxoid). IgG1 and IgG3 antibody predominated over IgG2 and IgG4 after primary immunization. Secondary immunization elicited enriched IgG1 and IgG4 responses. Primary immunization increased avidity indices of IgG to tetanus toxoid from approx. 0.9 (baseline) to a mean of 1.72 and secondary immunization significantly increased the avidity index to 2.56.

Juvenile periodontitis--a new perspective.

Juvenile periodontitis (JP) is a severe disease of the periodontium in adolescents. It is usually localized to the first permanent molars and (less commonly) the central incisors. The bacteria Actinobacillus actinomycetemcomitans (Aa) is currently implicated in the aetiology of JP since its numbers are high in JP pockets and low in subjects with healthy periodontal conditions or with adult periodontitis. However, Aa harvested from JP pockets and transferred to healthy sites in the same mouth are unable to colonize these areas or initiate disease (17). The conflicting evidence implicating intrinsic or induced impairment of host defence is reviewed. It is hypothesised that JP lesions are primarily of endodontic origin. By-products of an inflammatory process in the pulp enter the periodontium via dentinal tubules, lateral or furcation canals and drain through the periodontium into the mouth. The environmental conditions of the sinus select for bacteria such as Aa which secondarily infect the site and exacerbate the clinical situation by their potent virulence factors. Localized deep defects involving only one side of an interproximal space in an otherwise periodontally healthy mouth result. Studies of the pulpal status of JP teeth are indicated.

Endothelial cytotoxicity of Actinobacillus pleuropneumoniae.

The cytotoxicity of Actinobacillus pleuropneumoniae serotype 1 strain CM5 for porcine and bovine endothelial cells in vitro, was dose-dependent. This strain and its attenuated and avirulent substrain CM5A were equally cytotoxic. The cytotoxicity observed during five hours of exposure of endothelial cells to bacterial products was abolished if the bacteria were inactivated by heat or sonication. Exposure of the endothelial cells for five hours to 100 and 200 micrograms of purified lipopolysaccharide resulted in a partial cytotoxicity only, which was not enhanced in the presence of fresh guinea pig serum. The cytotoxicity of viable bacteria could be neutralised by a polyclonal rabbit antiserum to the purified 104kD haemolysin. A bacteria-free supernate of a culture of strain CM5 had both haemolytic and cytotoxic activity. The haemolytic activity could be neutralised completely by the anti-serum to the 104kD haemolysin, whereas the cytotoxic activity was only partially neutralisable. Hence A pleuropneumoniae is cytotoxic for endothelial cells and this cytotoxicity is possibly mediated by the 104kD haemolysin.

Actinobacillus species and their role in animal disease.

Actinobacillus species are Gram-negative bacteria responsible for several quite distinct disease conditions of animals. The natural habitat of the organisms is primarily the upper respiratory tract and oral cavity. A. lignieresii is the cause of actinomycosis (wooden tongue) in cattle: a sporadic, insidiously-developing granulomatous infection. In sharp contrast is A. pleuropneumoniae which is responsible for a rapidly spreading often fatal pneumonia, common among intensively reared pigs. Detailed investigation of this organism has provided a much clearer picture of the bacterial factors involved in causing disease. A. equuli similarly causes a potent septicaemia in the neonatal foal; growing apparently unrestricted once infection occurs. Other members of the genus induce characteristic pathogenesis in their preferred host, with one, A. actinomycetemcomitans, being a cause of human periodontal disease. This article reviews recent understanding of the taxonomy and bacteriology of the organisms, and the aetiology, pathogenicity, diagnosis and control of animal disease caused by Actinobacillus species.

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.

Actinobacillus pleuropneumoniae: molecular aspects of virulence and pulmonary injury.

Contributions made by several laboratories in the area of Actinobacillus pleuropneumoniae virulence and its relationship to pulmonary disease will be reviewed briefly. Lung injury and subsequent disease, after infection with A. pleuropneumoniae, can be related to various bacterial toxins and host factors. Similar to other gram-negative bacteria. A. pleuropneumoniae has cell wall lipopolysaccharides which have been incriminated in a wide variety of toxic and tissue damaging processes. Virulent isolates of A. pleuropneumoniae have been shown to have a thick capsule whereas some avirulent isolates have a thin and easily removed capsule. The capsule of A. pleuropneumoniae is a linear unbranched polysaccharide composed of repeating dissaccharide subunits that bestow antiphagocytic properties to the bacterium but are also immunogenic. In addition, A. pleuropneumoniae has several chemically defined exotoxins. These toxins have generally been shown to be proteinaceous molecules that are hemolytic, cytotoxic, or edemogenic. Some of these toxins are proteolytic and others have the putative activity of being lytic for secretory IgA. Several of these molecules are capable of inducing lesions that are similar to those observed in natural infections and disease. Endogenous host factors have also been implicated in the development of lung lesions after infection by A. pleuropneumoniae Coagulation and inflammatory pathways have been demonstrated to be pivotal in the early phases of lesion development. In addition, the immune status of the animal is clearly related to the severity and ultimate outcome of A. pleuropneumoniae infection. To adequately treat and prevent this disease, we must understand the distinguishable interactions that occur between the host and the various molecular virulence attributes of A. pleuropneumoniae.

Taxonomy and identification of periodontopathogenic bacteria and related species.

The tumultuous evolution of oral and periodontal microbiology has focussed the attention of many researchers on the necessity to clarify the taxonomic position and identification criteria of putative periodontopathogens, in order to elucidate the role played by each species in the pathogenesis of periodontal disease. Many of the most important periodontopathogens recently underwent a radical reclassification process that may create some confusion and certainly deserves an accurate analysis aimed at focussing the actual situation of these bacteria. The taxonomic evolution and identification criteria of species of the genera Bacteroides, Prevotella, Porphyromonas and Actinobacillus is here analyzed in detail to clarify this recent evolutive taxonomic process, and to explain the importance of molecular studies for both taxonomic and identification purposes in this field.

Characterization of an attenuated strain of Actinobacillus pleuropneumoniae, serotype 1.

Pleuropneumonia is an important disease of swine caused by Actinobacillus pleuropneumoniae. Putative virulence determinants include capsule, lipopolysaccharide, and cytotoxin. We studied the virulence and virulence determinants of 2 strains: CM5 and CM5A of serotype 1. Strain CM5 was isolated from a pig with pleuropneumonia and passaged once in vitro; strain CM5A was a substrain of CM5 passaged 70 times in vitro. Pigs challenge exposed to an aerosol of 1.3 x 10(7) colony-forming units of CM5/ml died within 30 hours; pigs challenge exposed to an aerosol of 1.6 x 10(8) colony-forming units of CM5A/ml survived. The average thickness of the capsular layer was 137 nm in strain CM5 and 53 nm in strain CM5A in bacteria treated with homologous antibody and examined by transmission electron microscopy. Similarly, capsular material binding polycationic ferritin was found in colonies of strain CM5, but not in strain CM5A. The ratio of hexosamine to protein in extracted capsule of CM5 was more than twice that of CM5A. The polyacrylamide gel electrophoretic profile of the lipopolysaccharide, outer membrane proteins, and whole cell proteins did not differ between the 2 strains. Also, the amount of cytotoxin or endotoxin produced by the 2 strains during the logarithmic growth phase was not different. The electrophoretic profile of restriction endonuclease digested DNA was similar, with the exception of bands in the 750- and 620-basepair regions. It was concluded that attenuation of strain CM5A during in vitro passage was a result of reduced capsule production and that encapsulation is an important virulence determinant of A pleuropneumoniae, serotype 1.

The comparative pathogenicity of four serovars of Actinobacillus (Haemophilus) pleuropneumoniae.

The pathogenicity of 2 isolates of each of serovars 7, 3, 1 and 2 of Actinobacillus pleuropneumoniae was tested by intranasal inoculation into 60, 6-week-old large white pigs. Four dose rates varying from 0.27 to 560 x 10(6) organisms per pig with 10-fold serial dilutions were used. Surviving pigs were necropsied 7 days after inoculation. The proportion of pigs dying and developing gross lesions following infection was significantly greater for pigs given serotype 1 than for each of the other 3 serotypes, which did not differ significantly from each other. Twelve of 16 pigs given either of the 2 isolates of serovar 1 died after acute illness and 1 of 44 pigs given either of the 2 isolates each of serovars 7, 3 and 2 died. Pigs given serovar 1 showed high temperatures, severe respiratory distress, frothy haemorrhagic nasal discharge and weight loss. Lung lesions were produced in all 16 pigs given serovar 1, in 7 of 14 pigs given serovar 7, 7 of 14 pigs receiving serovar 3 and in 5 of 16 pigs given serovar 2. The lethal infections were characterised by a severe acute fibrinohaemorrhagic necrotising pleuropneumonia, whereas non-lethal cases had lung lesions ranging from necrotising purulent pleuropneumonia to abscessation. Significant differences between isolates in proportions of tissues culture positive for A. pleuropneumoniae for serovars 7 and 2, but not for serovars 3 and 1 suggested that isolates may vary in virulence within serovars, but more detailed studies are needed to clarify this point.

[Clinical, bacteriological, and immunological examination and treatment of two Papillon-Lefevre syndrome patients].

Papillon-Lefèvre Syndrome (PLS) is a rare disease accompanied by palmo-plantar hyperkeratosis and rapidly progressive periodontal breakdown of deciduous and permanent dentition. Two unrelated female PLS patients, four and seven years old, with severe periodontal destruction were examined. Antibody titers against seven strains by the enzyme-linked immunosorbent assay (ELISA), microbial cultures from deep periodontal pockets and mouth rinse samples and immunoblotting analysis were performed. Titers against Actinobacillus actinomycetemcomitans (A.a.) were found to be high by the ELISA test. Microbial cultures of A. a. were found in high percentage and immunoblotting results against sonicated extracts of an A. a. Y4 strain had similar patterns. All deciduous teeth were extracted from the younger patient, later permanent dentition erupted uneventfully and A. a. colonies could not be detected. However, the older patient did not exhibit improvement with conventional periodontal therapy and antibiotic (minocycline/erythromycin) treatment and A. a. colonies could be consistently cultured. After a subsequent ofloxacin medication, A. a. colony detection was suppressed. Furthermore, a reduction of gingival inflammation and pocket depth were observed. Therefore, A. a. was associated as an important pathogen in the etiology of periodontal disease in these PLS patients.