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Characterization of polymorphic mononuclear cell in porcine Actinobacillus pleuropneumonia.

The polymorphic mononuclear cells, arranged in whorling or palisading pattern, were usually found in the lung of Actinobacillus pleuropneumoniae-infected pigs. In order to understand the origin and characteristics of these cells, specific-pathogen free pigs were intratracheally inoculated with Actinobacillus pleuropneumoniae at a concentration of 5 x 10(6) CFU, then, sacrificed at 6, 12, 24 and 48 hours later. The cells in the alveolar spaces were observed with light and electron microscope, and cytochemically analyzed for acid phosphatase, alkaline phosphatase, alpha-naphthyl acetate esterase, and Sudan black B stains respectively. The results revealed that a lot of neutrophils were observed in the alveolar spaces at the early stage after inoculation. Twenty four hours later, polymorphic mononuclear cells abundantly appeared. Enzyme cytochemical findings indicated that some of the polymorphic mononuclear cells were macrophages, in which, acid phosphatase and alpha-naphthyl acetate esterase were detected, and others were type II pneumocytes which were positively stained with alkaline phosphatase and Sudan black B. Ultrastructural observation found that many lysosomes appeared in the macrophages' cytoplasm, and type II pneumocyte contained many lamellar bodies. Conclusively, it could be suggested that the polymorphic mononuclear cells were derived from macrophages and type II pneumocytes by cytochemical and electron microscopic examinations.

Acid Phosphatase↗

Leukotoxicity of an extract from Actinobacillus actinomycetemcomitans for human gingival polymorphonuclear leukocytes.

A soluble extract from Actinobacillus actinomycetemcomitans (designated strain Y4) caused dose-dependent cytotoxic changes in PMN isolated from the gingival crevices (C-PMN) of normal adults. When the toxin was preincubated with sera from patients with juvenile periodontitis, there was a significant inhibition of toxic activity. In contrast a variety of other sera from normal subjects with healthy gingiva, and from patients with chronic gingivitis, chronic periodontitis, recurrent herpes labialis, rheumatoid arthritis, or ulcerative colitis enhanced the leukotoxic activity. The neutralization of toxin by serum from patients with juvenile periodontitis was probably due to specific antibodies. Since Actinobacillus actinomycetemcomitans organisms can be frequently identified in subgingival plaque from patients with juvenile periodontitis, the capacity of Y4 toxin to kill C-PMN may contribute to the pathogenesis of this disease.

Actinobacillus Infections↗

Endocarditis caused by Actinobacillus actinomycetemcomitans.

The first two cases of Actinobacillus actinomycetemcomitans endocarditis in Israel are reported. One involved a 67 year old woman with mitral insufficiency and the other, a 48 year old alcoholic male with bioprosthetic mitral valve. The organism grew in three of seven and seven of fourteen blood culture specimens, taken during the first two or three days of hospitalization respectively. Blood cultures became positive after incubation for seven days. The first patient recovered after therapy with penicillin and an aminoglycoside, and the second one is still being treated. Previously described cases of Actinobacillus actinomycetemcomitans endocarditis are reviewed.

Actinobacillus Infections↗

Concurrent infection with Mycobacterium farcinogenes and Actinobacillus lignièresi in slaughtered cattle.

Concurrent infection with Actinobacillus lignieresii and Mycobacterium farcinogenes was detected in slaughtered cattle. The lesions were confined to the retropharyngeal, mandibular and parotid lymph nodes. The actinobacillus granulomas were seen either inside the caseous lesions or between the caseous part and the epithelioid zone of bovine farcy granuloma. In concomitant infections, actinobacillosis was always in the early active phase, whereas in actinobacillosis alone, active as well as healed stages were encountered.

Actinobacillosis↗

Pericarditis, pneumonia and brain abscess due to a combined Actinomyces--Actinobacillus actinomycetemcomitans infection.

A patient is described in whom there developed a pulmonary infiltrate and constrictive pericarditis caused by a combined Actinomyces and Actinobacillus actinomycetemcomitans infection, presumably originating from his poor dentition. The diagnosis was only made following repeated thoracotomy. After surgery, long-term treatment with antibiotics led to complete clinical recovery. None the less, some months later he was found to have a brain abscess which resolved during a further course of antibiotics. The variable clinical picture of actinomycosis is discussed, as well as the role of other bacteria frequently associated with actinomycotic infection, in particular Actinobacillus actinomycetemcomitans. The therapeutic implications are described.

Actinobacillus Infections↗

Construction of Actinobacillus pleuropneumoniae-Escherichia coli shuttle vectors: expression of antibiotic-resistance genes.

We constructed several cloning vectors, designated pGZRS-18/19 and pGZRS-38/39, which were based on an endogenous Actinobacillus pleuropneumoniae (Apl) 4.3-kb plasmid. They carry the lacZ alpha-complementation fragment and MCS from pUC18/19, and either the bla gene under the control of a putative Apl promoter or the KmR gene from Tn903. These vectors replicate in representative strains of Apl serotypes 1 and 7, Escherichia coli, Pasteurella haemolytica (Ph) and Haemophilus (Actinobacillus) actinomycetemcomitans. We also found that Apl and Ph did not express genes under the control of the lacZ or bla promoters, suggesting that their RNA polymerases may not utilize these promoters.

Actinobacillus pleuropneumoniae↗

Construction of pYGK, an Actinobacillus actinomycetemcomitans-Escherichia coli shuttle vector.

A shuttle vector that is capable of replicating in Actinobacillus actinomycetemcomitans (Aa) and Escherichia coli (Ec) was constructed by modifying the Actinobacillus pleuropneumoniae (Ap) plasmid pYG53. A DNA fragment containing the KmR gene was inserted into pYG53 to generate pYGK, which confers resistance to kanamycin in both Aa and Ec. By electroporation, Ec DH5alpha and 17 strains of Aa were transformed with pYGK with efficiencies ranging from 0.5 to 3 X 10(6) colonies per microgram of DNA. Plasmid pYGK exists at approx. 3-4 copies per cell in Ec. This plasmid will facilitate the genetic manipulation of Aa strains and the molecular analysis of virulence factors expressed by this organism.

Actinobacillus↗

Actinobacillus ureae meningitis: case report and review of the literature.

Actinobacillus urea, formerly known as Pasteurella ureae, is an uncommon commensal of the upper respiratory tract in humans. It has been identified as the primary pathogen in 10 cases of meningitis and several cases of pneumonia, sepsis, and peritonitis. A case is presented that represents another documented case of meningitis due to this rare organism. Risk factors associated with serious infection due to Actinobacillus ureae and basic management approaches to posttraumatic meningitis in general are discussed.

Actinobacillus Infections↗

Iron acquisition by Actinobacillus suis: identification and characterization of a single-component haemoglobin receptor and encoding gene.

Actinobacillus suis is an important swine pathogen. As with other pathogens, the ability of A. suis to acquire iron within the host is crucial for virulence. Here, we investigated the ability of seven strains of A. suis to acquire iron from haemoglobins. In growth assays, all strains could use porcine, bovine and human haemoglobins as iron sources for growth. Using solid phase binding assays, membranes derived from all strains, grown under iron-restricted conditions, were shown to bind all three haemoglobins. Competition binding assays indicated that these haemoglobins were bound by the same receptor and an affinity procedure allowed the isolation and identification of an iron-repressible, haemoglobin-binding polypeptide (approximately 105 kDa) from all strains. Nucleotide sequence analyses revealed that A. suis possesses a gene (hgbA) that encodes a homologue of the Actinobacillus pleuropneumoniae haemoglobin-binding protein, HgbA. hgbA, encoding a mature protein of 105 kDa, was shown to be preceded by a hugZ homologue; putative promoter sequences and a putative Fur box were located upstream of hugZ and RT-PCR revealed that hugZ and hgbA are co-transcribed and iron-repressible. It is concluded that the acquisition of haemoglobin-bound iron by A. suis involves a single-component receptor that is up-regulated in response to iron restriction.

Actinobacillus suis↗

Construction of a shuttle vector for the overexpression of recombinant proteins in Actinobacillus succinogenes.

To express foreign proteins in Actinobacillus succinogenes, a shuttle vector was constructed based on the Actinobacillus pleuropneumoniae-Escherichia coli shuttle vector, pGZRS-19. We demonstrated that A. succinogenes is transformed by electroporation at reasonably high efficiency, that pGZRS-19 is stable in A. succinogenes, and that the ampicillin resistance gene carried by pGZRS-19 is expressed in A. succinogenes. Three steps were then required to develop our A. succinogenes-E. coli shuttle vector. (i) The constitutively expressed A. succinogenes phosphoenolpyruvate carboxykinase gene, pckA, was cloned and sequenced. (ii) Its promoter region and ribosome-binding site were subcloned into pGZRS-19. (iii) Finally, the ColE1 origin of replication was added to the vector to increase its stability in E. coli. High levels of A. succinogenes phosphoenolpyruvate carboxykinase, E. coli NADP-dependent malic enzyme, and Bacillus subtilis NAD-dependent malic enzyme activities detected in recombinant A. succinogenes strains confirmed that A. succinogenes and foreign proteins could be expressed in A. succinogenes under control of the A. succinogenes pckA promoter carried by pLGZ920. A. succinogenes is sensitive to chloramphenicol and tetracycline. Although not expressed from their own promoters, the Tn9 chloramphenicol and the Tn10 tetracycline resistance genes are expressed under control of the pckA promoter, and they can be used as additional selection markers in A. succinogenes.

Actinobacillus↗

Distribution of RTX toxin genes in strains of [Actinobacillus] rossii and [Pasteurella] mairii.

Strains of [Actinobacillus] rossii, [Pasteurella] mairii and [Pasteurella] aerogenes can be isolated from abortion in swine. The RTX toxin Pax has previously been found only in those [P.] aerogenes strains isolated from abortion. Nothing is known about RTX toxins in field isolates of the other two species. To gain insight into the distribution of selected RTX toxin genes and their association with abortion, PCR screening for the pax, apxII and apxIII operons on 21 [A.] rossii and seven [P.] mairii isolates was done. Since species can be phenotypically misidentified, the study was backed up by a phylogenetic analysis of all strains based on 16S rRNA, rpoB and infB genes. The pax gene was detected in all [P.] mairii but not in [A.] rossii strains. No apx genes were found in [P.] mairii but different gene combinations for apx were detected in [A.] rossii strains. Most of these strains were positive for apxIII, either alone or in combination with apxII. Whereas pax was found to be associated to strains from abortion no such indication could be found with apx in [A.] rossii strains. Phylogenetically [A.] rossii strains formed a heterogeneous cluster separated from Actinobacillus sensu stricto. [P.] mairii strains clustered with [P.] aerogenes but forming a separate branch. The fact that [P.] aerogenes, [P.] mairii and [A.] rossii can phylogenetically clearly be identified and might contain distinct RTX toxin genes allows their proper diagnosis and will further help to investigate their role as pathogens.

Actinobacillus↗

Cloning and characterisation of type 4 fimbrial genes from Actinobacillus pleuropneumoniae.

Actinobacillus pleuropneumoniae is the cause of porcine pleuropneumoniae. Little is known about the mechanisms by which A. pleuropneumoniae colonises the respiratory tract. Fimbriae are common mediators of bacterial adherence to mucosal epithelia and have been observed on the surface of A. pleuropneumoniae cells. Here we report the identification and characterisation of the type 4 fimbrial structural gene (apfA) from A. pleuropneumoniae. In addition a number of open reading frames were identified in A. pleuropneumoniae that have significant homology to type 4 fimbrial biogenesis genes from other species, including a putative leader specific peptidase (apfD). A. pleuropneumoniae apfA codes for a predicted polypeptide of approximately 16kDa, removal of the leader sequence at the predicted cleavage site would yield a 14.5kDa polypeptide. The first 30 residues of the mature polypeptide are well conserved with other members of the group A type 4 fimbriae family. The signal sequence of ApfA is 13 amino acids in length and, unusually, the residue that precedes the cleavage site is alanine rather than glycine which is found in most other type 4 fimbriae. The C-terminus of ApfA possesses cysteine residues that are conserved in type 4 fimbriae of many species. In other type 4 fimbriae the distal C-terminal cysteines form a disulphide bond that produces a loop, which is important for the function of fimbriae and also comprises a major antigenic determinant. A motif within the predicted loop in ApfA was found to be highly conserved in type 4 fimbriae of other HAP organisms (Haemophilus, Actinobacillus, Pasteurella). The A. pleuropneumoniae type 4 fimbrial biogenesis genes showed the strongest homology to putative type 4 fimbrial genes of Haemophilus ducreyi. A. pleuropneumoniae apfA gene was shown to be present and highly conserved in different serotypes of A. pleuropneumoniae. Recombinant ApfA was produced and used to raise anti-ApfA antisera.

Actinobacillus pleuropneumoniae↗

Differentiation between major species of the Actinobacillus--Haemophilus--Pasteurella group by gas chromatography of trifluoroacetic acid anhydride derivatives from whole-cell methanolysates.

A method based on whole-cell methanolysis and trifluoroacetic acid anhydride derivatization was developed for routine laboratory differentiation between isolates from the Actinobacillus--Haemophilus--Pasteurella group. All species, except Haemophilus aphrophilus, contained D-glycero-D-mannoheptose, although in varying concentrations. The distribution of this sugar could be used to distinguish H. aphrophilus from Actinobacillus actinomycetemcomitans, H. paraphrophilus, H. influenzae type b, Pasteurella haemolytica, P. multocida and P. ureae, and also H. influenzae type b from Pasteurellae. The pattern of major sugars in P. ureae and P. haemolytica resembled that of A. actinomycetemcomitans. Major fatty acids of the whole-cell methanolysates provided no basis of interspecies differentiation.

Acetic Anhydrides↗

Detection and subtyping of Actinobacillus pleuropneumoniae strains by PCR-RFLP analysis of the tbpA and tbpB genes.

A PCR-based procedure for detection and serotype identification of Actinobacillus pleuropneumoniae strains was developed and evaluated. The A. pleuropneumoniae tbpA and tbpB genes were used as targets for amplification of DNA fragments, with a pair of specific primers for each gene. Amplification with tbpA primers rendered a 2.8-kb PCR product from all 12 A. pleuropneumoniae reference strains as well as from Actinobacillus suis strain CCM 5586, while amplification of a 1.9-kb PCR product was observed when testing ten Haemophilus parasuis strains of different serovars. Amplification of the tbpB gene from A. pleuropneumoniae serotypes 1, 6, 8 and 12, and A. suis CCM 5586 rendered an identical 1.8-kb fragment, while from A. pleuropneumoniae serotypes 2, 3, 4, 7, 9, 10 and 11, and H. parasuis strains it produced a 1.7-kb fragment. No PCR amplification product was observed when examining strains of 19 other swine pathogens or closely related species. The minimal detection limit for whole-cell A. pleuropneumoniae templates was between 5-50 and 3 x 10(2)-3 x 10(3) CFU when tbpA and tbpB specific primers, respectively, were used. Restriction fragment length polymorphism (RFLP) analysis of the PCR-generated products rendered different patterns, easily allowing us to discriminate between A. pleuropneumoniae, H. parasuis and A. suis and, more importantly, to distinguish ten RFLP A. pleuropneumoniae groups (the highest discrimination reported so far for a PCR assay with A. pleuropneumoniae), in such a way that the only serotypes with profiles identical to each other were 4 to 11 and 7 to 9. Moreover, the PCR-RFLP analysis was assayed in 36 A. pleuropneumoniae field isolates and in porcine samples (lungs and nasal swabs from experimentally infected animals). In both cases the system proved to be very efficient in A. pleuropneumoniae identification and serotype discrimination.

Actinobacillus pleuropneumoniae↗

Cloning and characterization of the Actinobacillus pleuropneumoniae fur gene and its role in regulation of ApxI and AfuABC expression.

The ferric uptake regulation (fur) gene was cloned and characterized from Actinobacillus pleuropneumoniae and it exhibited 97% amino acid sequence identity to the Haemophilus ducrey fur gene. The flanking regions of the fur gene included an upstream putative flavodoxin (fldA) gene and a downstream possible transmembrane protein gene of unknown function. A single promoter was identified by 5' rapid amplification of cDNA ends (RACE), but there were no sequences homologous to an Escherichia coli Fur box in the 5' upstream sequence. The A. pleuropneumoniae fur clone complemented an E. coli fur deletion mutant. Transcriptional analysis of the divergent promoters of the A. pleuropneumoniae toxin I operon (apxICABD)--and the Actinobacillus ferric uptake operon (afuABC) showed that Fur and calcium together positively regulated the transcription of apxICABD while Fur was a repressor for afuABC. Hemolytic activity was significantly induced by iron and calcium and Fur appeared to act as an activator under high calcium conditions and as a repressor under low calcium conditions. A possible regulator-binding site was suggested by the properties of a point mutation in 33 bp upstream of the apxIC gene. This point mutation affected ApxI and Afu expression in response to iron, calcium, or Fur. These results provide further proof that calcium and the A. pleuropneumoniae Fur protein play a role in the expression of ApxI and Afu.

Actinobacillus pleuropneumoniae↗

Necrotizing pneumonia caused by mixed infection with Actinobacillus actinomycetemcomitans and Actinomyces israelii: case report and review.

Actinobacillus actinomycetemcomitans is an important cause of human pulmonary infections, either alone or with Actinomyces species. It may be critical to isolate Actinobacillus in patients with pulmonary infection for selection of an effective antimicrobial regimen. Clindamycin has superseded penicillin as the sole antimicrobial drug for anaerobic bacterial necrotizing pneumonia and abscess. In the case presented herein, therapy with clindamycin failed to halt worsening necrotizing pneumonia or to prevent hematogenous dissemination. After clindamycin-resistant A. actinomycetemcomitans in addition to Actinomyces israelii were isolated, the patient was treated with penicillin, ciprofloxacin, and cefazolin and was ultimately cured.

Actinobacillus Infections↗

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.

Actinobacillus↗

A numerical taxonomic study of Actinobacillus, Pasteurella and Yersinia.

A numerical taxonomic study of strains of Actinobacillus, Pasteurella and Yersinia, with some allied bacteria, showed 23 reasonably distinct groups. These fell into three major areas. Area A contained species of Actinobacillus and Pasteurella: A. suis, A. equuli, A. lignieresii, P. haemolytica biovar A, P. haemolytica biovar T, P. multocida, A. actinomycetemcomitans, 'P. bettii', 'A. seminis', P. ureae and P. aerogenes. Also included in A was a composite group of Pasteurella pneumotropica and P. gallinarum, together with unnamed groups referred to as 'BLG', 'Mair', 'Ross' and 'aer-2'. Area B contained species of Yersinia: Y. enterocolitica, Y. pseudotuberculosis, Y. pestis and a group 'ent-b' similar to Y. enterocolitica. Area C contained non-fermenting strains: Y. philomiragia, Moraxella anatipestifer and a miscellaneous group 'past-b'. There were also a small number of unnamed single strains.

Actinobacillus↗