Non-contiguous finished genome sequence of Ornithobacterium rhinotracheale strain H06-030791.

The Gram-negative, pleomorphic, rod-shaped bacterium Ornithobacterium rhinotracheale is a cause of pneumonia and airsacculitis in poultry. It is a member of the family Flavobacteriaceae of the phylum "Bacteroidetes". O. rhinotracheale strain H06-030791 was isolated from the lung of a turkey in North Carolina in 2006. Its genome consists of a circular chromosome of 2,319,034 bp in length with a total of 2243 protein-coding genes and nine RNA genes. Genome sequences are available for two additional strains of O. rhinotracheale, isolated in 1988 and 1995, the latter described in a companion genome report in this issue of SIGS. The genome sequence of O. rhinotracheale strain H06-030791, a more contemporary isolate, will be of value in establishing core and pan-genomes for O. rhinotracheale and elucidating its evolutionary history.

Complete genome sequence of Ornithobacterium rhinotracheale strain ORT-UMN 88.

Ornithobacterium rhinotracheale strain ORT-UMN 88 is a Gram-negative, pleomorphic, rod-shaped bacterium and an etiologic agent of pneumonia and airsacculitis in poultry. It is a member of the family Flavobacteriaceae of the phylum Bacteroidetes. O. rhinotracheale strain ORT-UMN 88 was isolated from the pneumonic lung of a turkey in 1995. It was the isolate first used to experimentally reproduce disease in turkeys and has since been the focus of investigations characterizing potential virulence factors of the bacterium. The genome of O. rhinotracheale strain ORT-UMN 88 consists of a circular chromosome of 2,397,867 bp with a total of 2300 protein-coding genes, nine RNA genes, and one noncoding RNA gene. A companion paper in this issue of SIGS reports the non-contiguous finished genome sequence of an additional strain of O. rhinotracheale, isolated in 2006.

Genomic and proteomic characterization of SuMu, a Mu-like bacteriophage infecting Haemophilus parasuis.

BACKGROUND: Haemophilus parasuis, the causative agent of Glässer's disease, is prevalent in swine herds and clinical signs associated with this disease are meningitis, polyserositis, polyarthritis, and bacterial pneumonia. Six to eight week old pigs in segregated early weaning herds are particularly susceptible to the disease. Insufficient colostral antibody at weaning or the mixing of pigs with heterologous virulent H. parasuis strains from other farm sources in the nursery or grower-finisher stage are considered to be factors for the outbreak of Glässer's disease. Previously, a Mu-like bacteriophage portal gene was detected in a virulent swine isolate of H. parasuis by nested polymerase chain reaction. Mu-like bacteriophages are related phyologenetically to enterobacteriophage Mu and are thought to carry virulence genes or to induce host expression of virulence genes. This study characterizes the Mu-like bacteriophage, named SuMu, isolated from a virulent H. parasuis isolate. RESULTS: Characterization was done by genomic comparison to enterobacteriophage Mu and proteomic identification of various homologs by mass spectrometry. This is the first report of isolation and characterization of this bacteriophage from the Myoviridae family, a double-stranded DNA bacteriophage with a contractile tail, from a virulent field isolate of H. parasuis. The genome size of bacteriophage SuMu was 37,151 bp. DNA sequencing revealed fifty five open reading frames, including twenty five homologs to Mu-like bacteriophage proteins: Nlp, phage transposase-C-terminal, COG2842, Gam-like protein, gp16, Mor, peptidoglycan recognition protein, gp29, gp30, gpG, gp32, gp34, gp36, gp37, gpL, phage tail tube protein, DNA circulation protein, gpP, gp45, gp46, gp47, COG3778, tail fiber protein gp37-C terminal, tail fiber assembly protein, and Com. The last open reading frame was homologous to IS1414. The G + C content of bacteriophage SuMu was 41.87% while its H. parasuis host genome's G + C content was 39.93%. Twenty protein homologs to bacteriophage proteins, including 15 structural proteins, one lysogeny-related and one lysis-related protein, and three DNA replication proteins were identified by mass spectrometry. One of the tail proteins, gp36, may be a virulence-related protein. CONCLUSIONS: Bacteriophage SuMu was characterized by genomic and proteomic methods and compared to enterobacteriophage Mu.

Comparison of Haemophilus parasuis reference strains and field isolates by using random amplified polymorphic DNA and protein profiles.

BACKGROUND: Haemophilus parasuis is the causative agent of Glässer's disease and is a pathogen of swine in high-health status herds. Reports on serotyping of field strains from outbreaks describe that approximately 30% of them are nontypeable and therefore cannot be traced. Molecular typing methods have been used as alternatives to serotyping. This study was done to compare random amplified polymorphic DNA (RAPD) profiles and whole cell protein (WCP) lysate profiles as methods for distinguishing H. parasuis reference strains and field isolates. RESULTS: The DNA and WCP lysate profiles of 15 reference strains and 31 field isolates of H. parasuis were analyzed using the Dice and neighbor joining algorithms. The results revealed unique and reproducible DNA and protein profiles among the reference strains and field isolates studied. Simpson's index of diversity showed significant discrimination between isolates when three 10 mer primers were combined for the RAPD method and also when both the RAPD and WCP lysate typing methods were combined. CONCLUSIONS: The RAPD profiles seen among the reference strains and field isolates did not appear to change over time which may reflect a lack of DNA mutations in the genes of the samples. The recent field isolates had different WCP lysate profiles than the reference strains, possibly because the number of passages of the type strains may affect their protein expression.

Detection of a bacteriophage gene encoding a Mu-like portal protein in Haemophilus parasuis reference strains and field isolates by nested polymerase chain reaction.

A nested polymerase chain reaction (nPCR) assay was developed to determine the presence of a gene encoding a bacteriophage Mu-like portal protein, gp29, in 15 reference strains and 31 field isolates of Haemophilus parasuis. Specific primers, based on the gene's sequence, were utilized. A majority of the virulent reference strains and field isolates tested harbored the gene. The results suggest that the nPCR technique described in the current report could serve as a tool for epidemiological studies of H. parasuis.

Ornithobacterium rhinotracheale North American field isolates express a hemolysin-like protein.

Ornithobacterium rhinotracheale is a gram-negative bacterium responsible for the sporadic outbreaks of airsacculitis in poultry, accounting for millions of dollars in losses to the poultry industry annually. Although the organism was originally classified as non-beta-hemolytic, recent North American field isolates of O. rhinotracheale obtained from pneumonic lungs and air sacs indicated hemolytic activity on blood agar plates upon extended incubation for 48 hr at room temperature in air after initial incubation at 37 C for 48 hr under 7.5% CO2. This report characterizes the beta-hemolytic activity of O. rhinotracheale isolates by using in vitro kinetic hemolysis assays with sheep red blood cells, western blotting with leukotoxin-specific monoclonal antibodies, and isobaric tagging and relative and absolute quantitative (iTRAQ) analysis of O. rhinotracheale outer membrane protein digest preparations. The kinetic analyses of the hemolytic activity with red blood cells indicated that the protein is a pore former. iTRAQ analysis with membrane preparations revealed four peptides with homology to Mannheimia haemolytica leukotoxin and two peptides with homology to Actinobacillus actinoacetemcomitans leukotoxin. This is the first report that North American field isolates of O. rhinotracheale may express a hemolysin-like activity.

Iron acquisition by Ornithobacterium rhinotracheale.

Ornithobacterium rhinotracheale (ORT) is an emerging respiratory pathogen of poultry in North America that is causing millions of dollars in economic losses to the poultry industry. Ornithobacterium rhinotracheale is associated with airsacculitis, pleuritis, pneumonia, and consolidation of lungs. Little is known about the molecular mechanisms of infection. In this study, the mechanism of iron acquisition by O. rhinotracheale was explored. O. rhinotracheale strains grown under iron deprivation in media containing 200 microM 2,2'-dipyridyl did not secrete siderophores as measured by the chrome azurol S (CAS) agar and CAS solution assays. Filter disks impregnated with various protein-bound iron compounds and inorganic iron salts of Fe(III) and Fe(II) placed on iron-restricted agar inoculated with a lawn of O. rhinotracheale supported growth from sheep and porcine hemoglobins, ovotransferrin, Fe(III), and Fe(II), but they did not support growth from bovine transferrin, bovine apo-transferrin, bovine lactoferrin, and hemin. However, both bovine hemoglobin and transferrin supported growth of O. rhinotracheale serotype C. Four immunoreactive proteins involved in iron acquisition were identified in an O. rhinotracheale membrane extract by using mass spectrometry. Furthermore, O. rhinotracheale field strains showed differential sensitivity to 2,2'-dipyridyl. Of the 72 field strains tested, 22 strains were resistant to the iron chelator at concentrations of 50 microM and 100 microM, suggesting this attribute may be related to disease-producing potential of these strains. This is the first report on the identification of the iron acquisition mechanism of O. rhinotracheale.

Identification of five outer membrane-associated proteins among cross-protective factor proteins of Pasteurella multocida.

Fowl cholera is caused by Pasteurella multocida serovars A:1, A:3, and A:4. The 39-kDa cross-protective factor protein and four other membrane proteins of the membrane proteome of P. multocida were identified. We determined that the 39-kDa cross-protective protein was Pasteurella lipoprotein B, or PlpB.