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Actinobacillus succinogenes sp. nov., a novel succinic-acid-producing strain from the bovine rumen.

Strain 130ZT was isolated from the bovine rumen. It is a facultatively anaerobic, pleomorphic, Gram-negative rod. It exhibits a 'Morse code' form of morphology, which is characteristic of the genus Actinobacillus. Strain 130ZT is a capnophilic, osmotolerant succinogen that utilizes a broad range of sugars. It accumulates high concentrations of succinic acid (> 70 g l-1). Strain 130ZT is positive for catalase, oxidase, alkaline phosphatase and beta-galactosidase, but does not produce indole or urease. Acid but no gas is produced from D-glucose and D-fructose. 16S rRNA sequence analysis places strain 130ZT within the family Pasteurellaceae; the most closely related members of the family Pasteurellaceae have 16S rRNA similarities of 95.5% or less with strain 130ZT. Strain 130ZT was compared with Actinobacillus lignieresii and the related Bisgaard Taxa 6 and 10. Based upon morphological and biochemical properties, strain 130ZT is most similar to members of the genus Actinobacillus within the family Pasteurellaceae. It is proposed that strain 130ZT be classified as a new species, Actinobacillus succinogenes. The type strain of Actinobacillus succinogenes sp. nov. is ATCC 55618T.

Actinobacillus↗

Isolation and characterization of some previously unreported taxa from poultry with phenotypical characters related to Actinobacillus-an Pasteurella species.

Cultural, morphologic, and biochemical characteristics of previously unreported taxa isolated from poultry and tentatively assigned to genus Actinobacillus Brumpt 1910 were compared to those of Actinobacillus lignieresii, A equuli, A. seminis, A. suis, avian haemolytic Actinobacillus sp., A.salpingitidis, avian Pasteurella haemolytica-like strains, P haemolytica biovar T, P, ureae. P. multocida, P. pneumotropica, P. gallinarum and P. anatipestifer. Evidence as obtained to indicate that taxon 1--3 was closely related to genus Actinobacillus Brumpt 1910, but sufficiently different from established species within that genus to constitute new distinct species. Taxon 4 had the cultural and biochemical characters of strains previously described by Clark and Godfrey. Strains designated P haemolytica-like could not be separated from A. salpingitidis on the basis of phenotypical characters. The final taxonomical position of taxon 1-4 in addition to strains designated avian haemolytic Actinobacillus sp. and P. haemolytica-like, however, has to await further taxonomical investigations including determination of mol % G + C in DNA and DNA hybridization, for which reason species names have been omitted.

Actinobacillus↗

Analysis of major antigens of Haemophilus (Actinobacillus) pleuropneumoniae and related organisms.

Outer membrane protein (OMP)-enriched extracts and whole-cell protein preparations of Haemophilus (Actinobacillus) pleuropneumoniae and related organisms were examined by polyacrylamide gel electrophoresis and immunoblotting. Both the OMP-enriched and whole-cell protein profiles of Actinobacillus suis, A. pleuropneumoniae (NAD-independent biovar), A. lignieresii, and Pasteurella haemolytica were very similar to those of H. pleuropneumoniae serotypes 1 to 8. Antisera prepared against H. pleuropneumoniae typically recognized three major OMP antigens with approximate molecular weights of 17,000 (17K), 32K, and 42K in immunoblots of H. pleuropneumoniae serotypes 1 to 8, Actinobacillus spp., and P. haemolytica. Antisera prepared against Actinobacillus spp. and Haemophilus sp. "minor group" also recognized these 17K, 32K, and 42K antigens. Using absorbed sera, we demonstrated that the 17K antigen had an epitope (or epitopes) common to all the gram-negative organisms examined, including Escherichia coli. The 32K and 42K antigens had epitopes common to members of the family Pasteurellaceae but, in the case of the 32K antigen, also contained unique epitopes. These results provide a basis for understanding the lack of specificity of serodiagnostic tests for H. pleuropneumoniae infection and provide another line of evidence for the association of H. pleuropneumoniae with the genus Actinobacillus.

Actinobacillus↗

Use of monoclonal antibodies to identify outer membrane antigens of Actinobacillus species.

Three monoclonal antibodies (LG17, LG30, and LG33) were used to identify outer membrane antigens of Actinobacillus sp (As8C isolate) cultured from the epididymides of an infected ram lamb. Specificity of the 3 antibodies to As8C antigens was determined by use of bacterial agglutination, the enzyme-linked immunosorbent assay, and the indirect fluorescent antibody test. Results of immunoelectron microscopy confirmed that each antibody was specific for epitopes on As8C outer membrane antigens. Evaluation by use of enzyme-linked immunoelectrotransfer blot indicated that target antigens for LG17 and LG33 antibodies had molecular weights of 10 kilodaltons and 43 kilodaltons, respectively. Multiple-band staining was observed with the LG33 antibody. The target antigen for the LG30 antibody could not be discerned by use of enzyme-linked immunoelectrotransfer blot. For each of the 3 monoclonal antibodies, enzyme-linked immunosorbent assay titers were obtained for Actinobacillus seminis, A actinomycetemcomitans, and 10 field isolates of Actinobacillus spp. Target antigens for LG17 and LG30 antibodies occurred infrequently or were absent on these bacteria. However, the target antigen for the LG33 antibody was shared by Actinobacillus seminis, A actinomycetemcomitans, and the 10 field isolates of Actinobacillus spp, indicating some diversity of outer membrane antigens between isolates.

Actinobacillus↗

Extracts of Prevotella intermedia and Actinobacillus actinomycetemcomitans inhibit alkaline phosphatase activity in osteoblastic cells in vitro.

In order to study the effects of sonicated extracts from Prevotella intermedia, Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, and other oral-related bacteria, as well as Escherichia coli on bone formation, clone MC3T3-E1 cells, which have retained osteoblastic activity, were cultured with various bacterial extracts. The addition of the sonicated extracts from Prevotella intermedia and Actinobacillus actinomycetemcomitans decreased the alkaline phosphatase activity in a dose-dependent fashion over the concentration range of 1-1000 ng ml-1 compared with the control. By contrast, the sonicated extracts from other oral bacteria including Porphyromonas gingivalis, Capnocytophaga ochracea, Streptococcus milleri and Streptococcus sanguis, and Escherichia coli did not decrease the alkaline phosphatase activity even in the presence of 100 ng ml-1 protein. The addition of Prevotella intermedia and Actinobacillus actinomycetemcomitans extracts that had been treated with heat and trypsin to the cell cultures also inhibited alkaline phosphatase activity in the cells, suggesting that inhibitory factors are not proteinaceous. Polymyxin B did not change the alkaline phosphatase activity in the cells treated with the extracts from Prevotella intermedia and Actinobacillus actinomycetemcomitans, suggesting that the inhibitory activity of the extracts is not lipopolysaccharide. The inhibitory effect of both extracts was observed in the molecular mass over 290 kDa eluted from Sephadex G-200 column. The inhibitory substances of Prevotella intermedia were partially purified and showed broad band with estimated molecular weight of 170-190 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These results indicate that Prevotella intermedia and Actinobacillus actinomycetemcomitans may play an important role in inhibiting bone formation as well as in stimulating bone resorption.

3T3 Cells↗

Increased frequency of isolation of Pasteurella and Actinobacillus species and related organisms.

Ninety-six clinical isolates of Pasteurella, Actinobacillus and related organisms were submitted to our reference laboratory for identification. The procedures for detecting the 11 identified species, Pasteurella multocida, Pasteurella haemolytica, Pasteurella ureae, Pasteurella pneumotropica, Actinobacillus lignieresii, Actinobacillus equuli, Actinobacillus actinomycetemcomitans, Haemophilus aphrophilus, Cardiobacterium hominis, and two unnamed species of CDC groups, HB-5 and EF-4, are described and their clinical importance is discussed. These organisms have been increasingly isolated in Japan and are most often associated with respiratory infections and endocarditis.

Actinobacillus↗

Neutrophil response, serum opsonic activity, and precipitating antibodies in human infection with Actinobacillus hominis.

Actinobacillus species are usually not considered as being human pathogens apart from A. actinomycetemcomitans. However, single cases of human meningitis, septicemia, and empyema caused by Actinobacillus lignieresii have been reported in the literature. This is the first reported case of Actinobacillus hominis giving rise to pleural-empyema in a patient with carcinoma of the lung. The function of peripheral blood neutrophils, serum opsonic activity and specific precipitating antibodies were investigated. Neutrophils from the patient exhibited an enhanced oxidative burst response measured by chemiluminescence assay. Furthermore, the opsonic activity of the serum from the patient was higher than that of a healthy control person. Several precipitating antibodies to various antigens of Actinobacillus hominis were demonstrated in the serum of the patient by crossed immunoelectrophoresis.

Actinobacillus↗

Actinobacillus spp. and related bacteria in infected wounds of humans bitten by horses and sheep.

We describe the isolation of Actinobacillus lignieresii and an A. equuli-like bacterium from an infected horse-bite wound in a 22-year-old stable foreman and A. suis from a bite injury in a 35-year-old man who had been attacked by a horse. A. lignieresii was also isolated in pure culture from an infected sheep-bite wound in a rural worker. These species of the genus Actinobacillus are primarily associated with animals and animal diseases and are rarely isolated from humans. The purpose of this report is to raise awareness of the possible occurrence of Actinobacillus spp. in bite wounds inflicted by farm animals and to discuss the difficulties encountered in the identification of species of Actinobacillus and related bacteria.

Actinobacillus↗

The induction of specific protection against Actinobacillus pleuropneumoniae infection by specific DLE.

Immunoprophylactic and immunotherapeutic effects of specific actinobacillus DLE (dialysable leukocyte extract) were studied in model pigs experimentally infected with Actinobacillus pleuropneumoniae. Specific DLE used for immunoprophylactic purposes was administered intramuscularly to pigs either in two doses in combination with one dose of actinobacillus vaccine (1st group) or in a single dose (2nd group). All animals of the two experimental groups survived the intranasal A. pleuropneumoniae challenge (2 x 2 ml 2-5 x 10(10) c.f.u.) while 3 of 5 animals of the control group died of haemorrhagic-necrotic fibrinous pneumonia. DLE administered to pigs for immunotherapeutic purposes was applied intravenously in two doses 1 and 5 h after the actinobacillus challenge (1st group), and in a single dose 5 h following the challenge (2nd group). All animals of the first group survived the challenge while 3 out of 5 animals of the second group and 4 out of 5 animals of the control group died during the experiment.

Actinobacillus Infections↗

Application of multivariate analyses of enzymic data to classification of members of the Actinobacillus-Haemophilus-Pasteurella group.

Outer membrane vesicles and fragments from Actinobacillus actinomycetemcomitans, Actinobacillus lignieresii, Actinobacillus ureae, Haemophilus aphrophilus, Haemophilus paraphrophilus, Haemophilus influenzae, Haemophilus parainfluenzae, Pasteurella haemolytica, and Pasteurella multocida were isolated and examined semiquantitatively for 19 enzyme activities by using the API ZYM micromethod. The enzyme contents of vesicles and fragments were compared with the enzyme contents of whole cells of the same organisms. Enzymic data were analyzed by using principal-component analysis and soft independent modeling of class analogy. This technique allowed us to distinguish among the closely related organisms A. actinomycetemcomitans, H. aphrophilus, and H. paraphrophilus. A. actinomycetemcomitans was divided into two groups of strains. A. lignieresii fell outside or on the border of the A. actinobacillus class. A. ureae, H. influenzae, H. parainfluenzae, P. haemolytica, and P. multocida fell outside the A. actinomycetemcomitans, H. aphrophilus, and H. paraphrophilus classes.

Actinobacillus↗

Reclassification of Actinobacillus actinomycetemcomitans, Haemophilus aphrophilus, Haemophilus paraphrophilus and Haemophilus segnis as Aggregatibacter actinomycetemcomitans gen. nov., comb. nov., Aggregatibacter aphrophilus comb. nov. and Aggregatibacter segnis comb. nov., and emended description of Aggregatibacter aphrophilus to include V factor-dependent and V factor-independent isolates.

The aim of this study was to reinvestigate the relationships and the generic affiliations of the species Actinobacillus actinomycetemcomitans, Haemophilus aphrophilus, Haemophilus paraphrophilus and Haemophilus segnis. The nicotinamide phosphoribosyltransferase gene (nadV) conferring V factor-independent growth was identified in Haemophilus aphrophilus. The gene encodes a polypeptide of 462 amino acids that shows 74.5 % amino acid sequence identity to the corresponding enzyme from Actinobacillus actinomycetemcomitans. Ten isolates of Haemophilus paraphrophilus all carried a nadV pseudogene. DNA from Haemophilus aphrophilus was able to transform Haemophilus paraphrophilus into the NAD-independent phenotype. The transformants carried a full-length nadV inserted in the former locus of the pseudogene. The DNA-DNA relatedness between the type strains of Haemophilus aphrophilus and Haemophilus paraphrophilus was 77 %. We conclude that the division into two species Haemophilus aphrophilus and Haemophilus paraphrophilus is not justified and that Haemophilus paraphrophilus should be considered a later heterotypic synonym of Haemophilus aphrophilus. Forty strains of Actinobacillus actinomycetemcomitans, Haemophilus aphrophilus and Haemophilus segnis were investigated by multilocus sequence analysis. The 40 strains form a monophyletic group clearly separate from other evolutionary lineages of the family Pasteurellaceae. We propose the transfer of Actinobacillus actinomycetemcomitans, Haemophilus aphrophilus and Haemophilus segnis to a new genus Aggregatibacter gen. nov. as Aggregatibacter actinomycetemcomitans comb. nov. (the type species; type strain ATCC 33384(T)=CCUG 13227(T)=CIP 52.106(T)=DSM 8324(T)=NCTC 9710(T)), Aggregatibacter aphrophilus comb. nov. (type strain ATCC 33389(T)=CCUG 3715(T)=CIP 70.73(T)=NCTC 5906(T)) and Aggregatibacter segnis comb. nov. (type strain HK316(T)=ATCC 33393(T)=CCUG 10787(T)=CCUG 12838(T)=CIP 103292(T)=NCTC 10977(T)). The species of the genus Aggregatibacter are independent of X factor and variably dependent on V factor for growth in vitro.

Actinobacillus↗

Serum neutralizing activity against Actinobacillus actinomycetemcomitans leukotoxin in juvenile periodontitis.

A relatively high incidence of infection by Actinobacillus actionomycetemcomitans can be shown in subgingival plaque samples obtained from patients with juvenile periodontitis. These organisms possess a potent leukotoxin(s) which rapidly destroys isolated human polymorphonuclear leukocytes (PMNs) and monocytes. If such leukotoxins operate in vivo, they could deprive the gingival crevice area of an essential antibacterial defense mechanism. We have found that sera from juvenile periodontitis patients consistently (greater than 90%) contain antibodies which neutralize Actinobacillus actinomycetemcomitans leukotoxin(s). On the other hand, sera from normal individuals or patients with other types of periodontal disease usually amplified rather than inhibited the leukotoxic reaction. Many patients with juvenile periodontitis have demonstrable defects in PMN or monocyte chemotaxis and this may place them at risk to gingival infection by Actinobacillus actinomycetemcomitans. The immune response against these organisms could be a crucial determinant in the course of juvenile periodontitis. While this disease is relatively rare, it does cause immeasurable emotional, physical and economic hardship for patients and their families. The identification of Actinobacillus actinomycetemcomitans as a potential pathogen in this disorder may eventually lead to specific forms of therapy to prevent and eliminate infection by this organism in these patients.

Actinobacillus↗

Cellular fatty acid and soluble protein composition of Actinobacillus actinomycetemcomitans and related organisms.

The cellular fatty acid and protein content of twenty-five representative strains of Actinobacillus actinomycetecomitans isolated from juvenile and adult periodontitis patients was compared to that of 15 reference strains of oral and nonoral Actinobacillus species and Haemophilus aphrophilus. Trimethylsilyl derivatives of the fatty acid methyl esters were analyzed by gas-liquid chromatography. The predominant fatty acids of all 40 strains examined were 14:0, 3-OH 14:0, 16 delta, and 16:0. Actinobacillus seminis (ATCC 15768) was unlike the other strains examined because of a greater amount of 14:0 detected. The soluble protein analysis using polyacrylamide gel electrophoresis revealed that A. actinomycetemcomitans, H. aphrophilus, and nonoral Actinobacillus species possessed distinct protein profiles attesting to the validity of separating these organisms into different species. Established biotypes of A. actinomycetemcomitans could not be differentiated on the basis of fatty acid or protein profiles.

Actinobacillus↗

Actinobacillus sp. bacteremia in foals: clinical signs and prognosis.

Medical records of 101 blood culture-confirmed bacteremic foals were reviewed to determine whether foals with Actinobacillus sp. bacteremia are affected at an earlier age, have more severe signs of disease, and have a worse prognosis than do foals with bacteremia of other causes. Thirty percent (30/101) of bacteremic foals had Actinobacillus sp. cultured, and these were 2 times more likely to die (crude odds ratio [OR(CR)] 0.8, 4; P = .14), with a survival rate of 43% (13/30) compared to the overall survival rate of 55% (56/101). When compared to other bacteremic foals, foals with actinobacillosis were 7 times more likely to have been sick from birth (adjusted odds ratio [OR(ADJ)] 2, 26; P = .003) and 6 times more likely to have diarrhea (OR(ADJ) 1, 22; P = .009). By bivariate analysis. foals with Actinobacillus sp. bacteremia were 5 times more likely to have a sepsis score >11 (OR(CR) 1, 18; P = .007), 6 times more likely to be obtunded (OR(CR) 2, 20; P = .005), and 3 times more likely to have pneumonia (OR(CR) 1, 7; P = .03). Furthermore, Actinobacillus sp. bacteremic foals were 27 times more likely to have a segmented neutrophil count <3.3 X 10(9) cells/L (OR(ADJ) 4, 166: P < .0001) and were 4.5 times more likely to have a band neutrophil count >0.46 x 10(9) cells/L (OR(ADJ) 1, 17; P = .02) when compared to foals that had bacteremia caused by either gram-negative enteric or gram-positive organisms. Sepsis score was < or = 11 in 49% (29/59) of bacteremia foals aged <13 days for which a discernible sepsis score was calculable. Results of this study should improve the diagnostic sensitivity of clinical examinations of neonatal foals, thereby facilitating treatment decisions.

Actinobacillosis↗

Tissue pharmacokinetics of florfenicol in pigs experimentally infected with Actinobacillus pleuropneumoniae.

UNLABELLED: The aim of this study has been to determine the tissue pharmacokinetic parameters of florfenicol in the pigs experimentally infected with Actinobacillus pleuropneumoniae. 21 crossed-bred (Duroc x Landrace x Yorkshire) local species of pigs were infected experimentally with Actinobacillus pleuropneumoniae serotype 1 and confirmed as typical sub-acute pleuropneumonia. A single dose of 20 mg/kg body weight of florfenicol, a novel animal-using antibiotic, was administrated intramuscularly in the pigs and then samples of blood, lung, trachea with bronchi, liver, kidney and muscle were taken at scheduled time points. Drug concentrations were determined by high performance liquid chromatography (HPLC) with an ultraviolet detector via extraction with ethyl acetate under nitrogen flow. The statistic moment theory (SMT) mathematic package was applied to calculate the tissue pharmacokinetic parameters of florfenicol in the infected model. AUC of lung, trachea with bronchi, liver, kidney and muscle were 121.69, 79.37, 81.05, 181.2, and 94.07 mg/l x h, respectively, MRT were from 34.66 to 90.17 h, and t1/2beta from 24.75 to 69.34 h, respectively. CONCLUSIONS: Florfenicol was widely distributed in these tissues and maintained the effective therapeutic concentrations especially in the respiratory tract tissues that are the target organs of Actinobacillus pneuropneumoniae. CLINICAL SIGNIFICANCE: Tissue pharmacokinetic data could be evidence for regime designing of florfenicol in treatment of porcine pleuropneumonia.

Actinobacillus Infections↗

Identification and detection of Actinobacillus pleuropneumoniae by PCR based on the gene apxIVA.

The apxIVA gene, a recently discovered RTX determinant of Actinobacillus pleuropneumoniae, was shown to be species-specific. DNA hybridization experiments using probes for various regions of apxIVA revealed that the 3'-terminus of this gene was present in all 14 serotypes of A. pleuropneumoniae but absent from phylogenetically related species. A primer pair spanning this region specifically amplified a 422bp fragment in PCR experiments with DNA from the reference strains of the 14 serotypes and 194 field strains isolated from various geographic locations worldwide. DNA sequence analysis of PCR products derived from all serotypes were identical except in serotypes 3, 8, and 10, which showed minor differences. The PCR did not amplify any product when DNA from 17 different bacterial species closely related to A. pleuropneumoniae was used as template. In addition, the PCR was negative with DNA of several Actinobacillus sp. which were initially characterized as A. pleuropneumoniae using routine phenotypic and serological analyses but which were subsequently shown by 16S rRNA sequence analysis to belong to yet undefined Actinobacillus species. The sensitivity of the PCR was determined to be 10pg of A. pleuropneumoniae DNA. A set of nested primers amplified a 377bp fragment specifically with A. pleuropneumoniae DNA. DNA titration experiments using the flanking and nested primer pairs showed an improved level of sensitivity to approximately 10fg of genomic DNA. The nested PCR was used to monitor the spread of A. pleuropneumoniae in pigs experimentally infected with a virulent serotype 1 strain and housed in a controlled environment facility. A. pleuropneumoniae DNA could be detected by nested PCR in nasal swab samples of infected pigs receiving either a high dose (5x10(5)) or a low dose (1x10(4)) challenge and in unchallenged cohorts that were contact-infected by the inoculated animals. Furthermore, PCR confirmed the presence of A. pleuropneumoniae in 16/17 homogenates from necrotic lung lesions, while the bacterium was successfully recovered from 13 of these lesions by culture.

Actinobacillus Infections↗

Evaluation of an indirect enzyme-linked immunosorbent assay (ELISA) for detection of antibodies to the Apx toxins of Actinobacillus pleuropneumoniae.

The reference strains of the 12 serotypes of Actinobacillus pleuropneumoniae express one or two of three different RTX exotoxins designated Apx I, Apx II and Apx III. The toxins are important virulence factors. In the present study, ELISAs with purified Apx I, Apx II and Apx III, respectively, as antigen were evaluated as candidates for serological diagnosis of Actinobacillus pleuropneumoniae infection in pigs. The pigs were inoculated with biotype 1, serotypes 1-12, and biotype 2, serotype 14, respectively. A strong humoral antibody response was seen to all the three antigens in most pigs irrespective of the serotype used for inoculation. However, titers to the exotoxins secreted by the serotype used for inoculation were generally highest. The results show that toxin proteins of Actinobacillus pleuropneumoniae are antigenically related and that a correlation between serotype and secretion of exotoxin is not revealed serologically in the ELISA test.

Actinobacillus Infections↗

Differentiation of Actinobacillus pleuropneumoniae strains by sequence analysis of 16S rDNA and ribosomal intergenic regions, and development of a species specific oligonucleotide for in situ detection.

The aims of this study were to characterize and determine intraspecies and interspecies relatedness of Actinobacillus pleuropneumoniae to Actinobacillus lignieresii and Actinobacillus suis by sequence analysis of the ribosomal operon and to find a species-specific area for in situ detection of A. pleuropneumoniae. Amplification and sequence analysis of the 16S-23S rDNA ribosomal intergenic sequence (RIS) from the three species showed the existence of two RIS's, differing by about 100 bp. Both sequences contained a region resembling the ribonuclease III cleavage site found in Escherichia coli. The smaller RIS contained a Glu-tRNA gene, and the larger one contained genes encoding Ile-tRNA and Ala-tRNA. These tRNA's showed a high sequence homology to the respective tRNA genes found in E. coli. Sequence analysis of the RIS's showed a high degree of genetic similarity of 24 strains of A. pleuropneumoniae. The larger RIS's were different between the 3 species tested. The sequence of the 16S ribosomal gene was determined for 8 serotypes of A. pleuropneumoniae. These sequences showed only minor base differences, indicating a close genetic relatedness of these serotypes within the species. An oligonucleotide DNA probe designed from the 16S rRNA gene sequence of A. pleuropneumoniae was specific for all strains of the target species and did not cross react with A. lignieresii, the closest known relative of A. pleuropneumoniae. This species-specific DNA probe labeled with fluorescein was used for in situ hybridization experiments to detect A. pleuropneumoniae in biopsies of diseased porcine lungs.

Actinobacillus Infections↗