Method for culturing Candidatus Ornithobacterium hominis.

Candidatus Ornithobacterium hominis has been detected in nasopharyngeal microbiota sequence data from around the world. This report provides the first description of culture conditions for isolating this bacterium. The availability of an easily reproducible culture method is expected to facilitate deeper understanding of the clinical significance of this species.

Phylogenetic relationship of Ornithobacterium rhinotracheale isolated from poultry and diverse avian hosts based on 16S rRNA and rpoB gene analyses.

BACKGROUND: Ornithobacterium (O.) rhinotracheale is an emerging bacterial pathogen in poultry and not fully understood to date. Because of its importance particularly for the global turkey meat industry, reliable diagnostic and characterization methods are needed for early treatment and in future for better vaccine production. The host range of birds infected by O. rhinotracheale or carrying the bacterium in their respiratory tract has constantly increased raising important epidemiological and taxonomic questions for a better understanding of its diversity, ecology and transmission cycles. The purpose of this study was to introduce partial rpoB gene sequencing for O. rhinotracheale into routine diagnostics to differentiate strains isolated from poultry and more diverse avian hosts (i.e., birds of prey, corvids and pigeons) and to compare phylogenetic relationships with results from 16S rRNA gene analysis and multilocus sequence typing (MLST). RESULTS: Partial 16S rRNA gene analysis revealed a high level of homogeneity among the 65 investigated O. rhinotracheale sequences with similarity values ranging from 98.6 to 100% between sequences from non-galliform and poultry species. The corresponding rpoB gene sequences were heterogeneous and ranged in their similarity values from 85.1 to 100%. The structure of the rpoB tree was in strong correlation with previous MLST results revealing three main clusters A (poultry and birds of prey), B (poultry, birds of prey and corvids) and C (pigeons), which were clearly separated from each other. CONCLUSIONS: By using partial sequences from a single gene, the rpoB gene analysis is in good agreement with MLST results with a slight decrease in resolution to distinguish more similar strains. The present results provide strong evidence that traditional phenotypic and genetic methods may not properly represent the heterogeneous group of bacteria classified as O. rhinotracheale. From housekeeping gene analyses, it is very likely that the genus Ornithobacterium includes additional species and partial rpoB gene sequencing can be recommended as fast, cost-effective and readily available method to identify strains and differentiate between O. rhinotracheale and Ornithobacterium-like bacteria.

'Candidatus Ornithobacterium hominis': insights gained from draft genomes obtained from nasopharyngeal swabs.

'Candidatus Ornithobacterium hominis' represents a new member of the Flavobacteriaceae detected in 16S rRNA gene surveys of people from South-East Asia, Africa and Australia. It frequently colonizes the infant nasopharynx at high proportional abundance, and we demonstrate its presence in 42 % of nasopharyngeal swabs from 12-month-old children in the Maela refugee camp in Thailand. The species, a Gram-negative bacillus, has not yet been cultured, but the cells can be identified in mixed samples by fluorescent hybridization. Here, we report seven genomes assembled from metagenomic data, two to improved draft standard. The genomes are approximately 1.9 Mb, sharing 62 % average amino acid identity with the only other member of the genus, the bird pathogen Ornithobacterium rhinotracheale. The draft genomes encode multiple antibiotic-resistance genes, competition factors, Flavobacterium johnsoniae-like gliding motility genes and a homologue of the Pasteurella multocida mitogenic toxin. Intra- and inter-host genome comparison suggests that colonization with this bacterium is both persistent and strain exclusive.

Phylogenetic relationship of Ornithobacterium rhinotracheale strains.

The bacterium Ornithobacterium rhinotracheale is associated with respiratory disease in wild birds and poultry. In this study, the phylogenetic analysis of nine reference strains of O. rhinotracheale belonging to serovars A to I, and eight Mexican isolates belonging to serovar A, was performed. The analysis was extended to include sequences from another 23 strains available in the public domain. The analysis showed that the 40 sequences formed six clusters, I to VI. All eight Mexican field isolates were placed in cluster I. One of the reference strains appears to present genetic diversity not previously recognized and was placed in a new genetic cluster. In conclusion, the phylogenetic analysis of O. rhinotracheale strains, based on the 16S rRNA gene, is a suitable tool for epidemiologic studies.

Molecular Characterization of the Recently Emerged Poultry Pathogen Ornithobacterium rhinotracheale by Multilocus Sequence Typing.

Ornithobacterium rhinotracheale (ORT) is an economically important bacterial pathogen of turkeys and chickens worldwide. Since its first detection, a variety of typing methods have been used to gain basic knowledge about the bacterial population structure, an issue that still needs to be addressed. Serological characterization revealed at least 18 different serotypes (A-R) with ORT of serotype A to be predominate among poultry. This study aimed to establish a multilocus sequence typing (MLST) scheme for ORT that could easily be used by other laboratories and allows for worldwide comparison of sequence data. For this purpose, 87 ORT strains from different poultry hosts, geographical origins, years of isolation and serotypes were included in the analysis to identify correlations. Fourteen different sequence types (ST) were found. The most common ST1 was identified in 40 ORT strains from turkeys and chickens on 4 continents and in 3 different European countries. Together with ST9, both STs represented over three quarters (77%) of ORT strains used in the MLST analysis and included strains of frequently cross-reacting ORT serotypes A, E and I. Nine STs were only represented by one ORT strain and might indicate possible avian host, disease or serotype-specific relationships. In contrast, discrepancies between serotype and phylogenetic relatedness were clearly demonstrated by ORT strains that belonged to identical serotypes but differed in their ST. The overall identified low genetic diversity among strains isolated from turkeys and chickens independent of host and geographical origins suggests that ORT has only recently been introduced into domestic poultry and dispersed worldwide.

Retrospective Study on the Isolation of Ornithobacterium rhinotracheale from Chickens and Turkeys in Central California: 294 cases (2000-12).

Ornithobacterium rhinotracheale is a rod-shaped, gram-negative, and mostly oxidase-positive bacterium that causes respiratory infections in chickens and turkeys worldwide and can also spread to nonrespiratory organs. The present report analyzes 294 cases in which O. rhinotracheale was isolated from turkeys or chickens in central California in the years 2000 through 2012. Two hundred sixteen cases were from turkey flocks and 78 from chicken flocks. The median age of turkey flocks was 8.7 wk; the median age of chicken flocks was 6.4 wk. From turkeys, O. rhinotracheale was more often isolated from August to January than during the rest of the year. Chickens cases were more evenly distributed throughout the year. The organs with the highest isolation rate were the infraorbital sinus and trachea, followed by lungs and air sacs. Isolation from other organs was rare. Pure cultures were obtained from relatively more turkey organs than chicken organs. The organ from which there was the highest chance to obtain a pure culture was the air sac. In 108 turkey flocks (50.0%) and 64 chicken flocks (82.1%) at least one other respiratory pathogen was detected. The most common gross lesions were increased mucus in trachea, caseous or fibrinous exudate in the air sacs, consolidated lungs indicating pneumonia, congested and edematous lungs, and a flattened trachea. For most types of lesions, the percentage of affected turkeys was higher than the percentage of affected chickens. The percentage of birds with lesions was higher if other respiratory pathogens were present. Overall, the host species (turkey or chicken) was a more important factor for the prevalence of most lesions than the detection of other respiratory pathogens. The most common histopathologic lesions in the sinus and trachea were heterophilic or mononuclear inflammatory cell infiltration. In the lungs and air sacs, the inflammation was characterized by heterophilic infiltration and/or fibrin accumulation. These results are helpful in selecting the most appropriate samples for isolation of O. rhinotracheale. In addition, they show the incidence of the bacterium in turkeys and chickens and which lesions can be expected after infection with O. rhinotracheale, and they indicate that in some cases O. rhinotracheale can be the primary, or at least the major, pathogen.

Detection and typing of Ornithobacterium rhinotracheale from German poultry flocks.

Ornithobacterium rhinotracheale (ORT) is a gram-negative staining rod. In chickens and turkeys ORT causes a respiratory disease. Between 2009 and 2011 some 714 dry swabs taken from diseased turkeys, broilers, broiler breeders, layers, or from unknown origin were investigated by PCR for the presence of ORT. Swabs that tested positive numbered 197 out of 481 from turkeys (41.0%), 10 out of 144 from broilers or broiler breeders (6.9%), 17 out of 28 from layers (60.7%), and 26 out of 61 from unknown origin (42.6%). The results of three swabs from turkeys were suspect. Furthermore, 310 isolates from turkeys and 62 isolates from unknown origin were typed using an agar gel precipitation (AGP) test. Of the isolates from turkeys, 56.1% belonged to serotype A and 20.6% to serotype E. The prevalence of other isolates was below 10%. Serotypes D, F, and K were not detected. Eleven isolates were not typable with reference sera against serotypes A-L. The three serotypes most often found in the isolates from unknown origin were A (35.5%), B (19.4%), and C (12.9%). The prevalence of other isolates was below 10%. Serotypes F and K were not detected. Seven isolates were not typable with reference sera A-L. Cross-reactions, especially of serotype A isolates with serotypes I, H, and J, were common. Additionally, the partial 16S ribosomal RNA (rRNA) and the complete Or01 genes of reference strains A-H and of nine field isolates were cloned and sequenced. Identity scores of 16S rRNA fragments were between 98% and 100%. Identities of the Or01 sequences were between 94% and 100%. Phylogenetic trees of both genes showed similarities. However, there was no apparent correlation between reference strains and isolates belonging to one serotype, so sequencing of 16S rRNA or of the Or01 gene does not seem to be a suitable method to replace the AGP for serotyping. Further investigations are necessary to clarify the cross-reactions between different serotypes and their real role in the pathogenicity and in consideration of vaccine production.

Co-infection of Ornithobacterium rhinotracheale with Streptococcus zooepidemicus in chickens.

An outbreak of bronchial embolization with 50%-70% morbidity and 30% mortality occurred in broilers in northeast China. This highly contagious disease is characterized by the sudden onset of clinical symptoms, including dyspnea, hemorrhagic tracheal discharge, and bronchial obstruction. Subsequently, six strains of Ornithobacterium rhinotracheale (ORT) and three strains of Streptococcus zooepidemicus were isolated from the various organs and identified using biochemical tests and PCR methods. The pathogenesis of embolization in chickens is poorly understood. The current experimental study confirmed that ORT infection alone could induce a significantly fatal hemorrhagic pneumonia and high mortality in comparison with S. zooepidemicus infection. Moreover, co-infection of ORT with S. zooepidemicus could induce even higher mortality, with severe bronchial obstruction, than that observed in chickens infected with S. zooepidemicus or ORT alone. Therefore, the combination of ORT and S. zooepidemicus may be associated with the outbreak of chicken bronchial embolization. Further investigation of the pathogenesis of ORT and Streptococcus is urgently needed.

Use of random amplified polymorphic DNA analysis and single-enzyme amplified fragment length polymorphism in molecular typing of Ornithobacterium rhinotracheale strains.

Ornithobacterium rhinotracheale (ORT) is a bacterium common to commercial poultry and wild birds throughout the world. It is also known as a causative agent of respiratory diseases. A total of 93 ORT isolates originating from chickens, pigeons, ostriches, quail, turkeys, and an Asian crested goshawk (Accipiter trivirgatus) in Taiwan, between 2004 and 2006, were used in this study. High genetic similarity (97%-100%) in 16S rRNA sequence was revealed among the 50 randomly selected isolates, in addition to a reference strain (ATCC-51464) and seven reference sequences from GenBank. In order to obtain a greater genetic discrimination among the ORT isolates, random amplified polymorphic DNA (RAPD) and single-enzyme amplified fragment length polymorphism (SE-AFLP) methods were further conducted. The results showed that both RAPD and SE-AFLP assays showed higher discriminatory abilities than the 16S rRNA sequence assay. Genetic clustering revealed that chicken- and quail-origin isolates were genetically distinct from those of the ostrich, pigeon, and Asian crested goshawk-origin isolates. However, among the two typing methods, the turkey-origin isolates showed diverse genetic characteristics to domestic avian species. With this information, ecologic and epidemiologic studies could be furthered for the reduction and control of ORT transmission in Taiwan.

Hemagglutinating activity of serovar reference strains of Ornithobacterium rhinotracheale.

In the present study, the hemagglutinating activity of 9 reference strains (serovars A-I) of Ornithobacterium rhinotracheale was investigated by using fresh erythrocytes from 15 different species: chicken (broiler, rooster, hen), turkey, pigeon, quail, duck, Harris hawk (Parabuteo unicinctus), house finch (Carpodacus mexicanus), cow, sheep, horse, dog, rabbit, pig, human (groups A, B, AB, and O), and rainbow trout (Oncorhynchus mykiss). All 9 strains agglutinated rabbit erythrocytes. None of the strains was able to agglutinate hen, cow, horse, or rainbow trout erythrocytes. The number of positive reactions among the remaining species varied. Results indicate that the use of rabbit erythrocytes is better suited for testing the hemagglutinating activity of O. rhinotracheale.

Seroprevalence and identification of Ornithobacterium rhinotracheale from broiler and broiler breeder flocks in Thailand.

Ornithobacteriosis is an infectious disease of avian species that has been reported in almost all countries around the world, except Thailand. The objectives of this study were to determine the seroprevalence of Ornithobacterium rhinotracheale (ORT) and to isolate and identify ORT in broilers and broiler breeders in Thailand. Chicken antibodies had been randomly checked from 17 farms (19 flocks) of broilers and 23 farms (28 flocks) of broiler breeders. The seropositive flocks were 63% and 100% in broilers and broiler breeders, respectively. The sera analysis showed that the individual 280 broiler sera antibody responses were 67.5% negative, 12.9% suspect, and 19.6% positive. The individual antibody responses of 510 broiler breeder sera revealed 12.2% negative, 38.0% suspect, and 49.8% positive samples. The bacteria were isolated and identified by polymerase chain reaction (PCR). Bacterial isolation and identification revealed that nine isolates of the 12 PCR analysis samples showed positive results to PCR analysis. All the positive PCR samples were collected from the broiler breeder farms.

Application of polymerase chain reaction fingerprinting to differentiate Ornithobacterium rhinotracheale isolates.

Ornithobacterium rhinotracheale (ORT) is an infectious respiratory pathogen of chickens, turkeys, and wild birds. There are 18 serotypes of ORT reported worldwide. In this study, enterobacterial repetitive intergenic consensus (ERIC) polymerase chain reaction and random amplified polymorphic DNA assay with Universal M13 primer-based fingerprinting techniques were investigated for their ability to differentiate ORT isolates. The authors examined 50 field isolates and 8 reference strains of ORT for their genetic differences. The fingerprint patterns were compared with serotyping results of ORT by the agar gel precipitation test. M13 fingerprinting revealed different patterns for 6 reference serotypes of ORT that were tested, namely, C, D, E, I, J, and K. Ornithobacterium rhinotracheale reference serotypes A and F yielded indistinguishable fingerprints with M13 fingerprinting. The ERIC 1R technique discerned only 5 of the 8 reference serotypes of ORT. Distinct fingerprints were also found within the ORT serotypes with both techniques. From 58 isolates of ORT that were fingerprinted belonging to 8 ORT serotypes, 10 different fingerprints were obtained with M13 fingerprinting and 6 different fingerprints were obtained with ERIC 1R fingerprinting. M13 fingerprinting technique was found to be more discriminative in differentiating ORT isolates than the ERIC 1R fingerprinting technique. These results suggest that fingerprinting techniques may be a more discerning tool for characterizing ORT isolates than the serological test using the agar gel precipitation test. This fingerprinting technique could potentially be a valuable tool in identifying an isolate from a clinical outbreak of ORT infection for development of an autogenous vaccine.

Isolation and characterization of Ornithobacterium rhinotracheale from chickens in Brazil.

Ornithobacterium rhinotracheale (ORT) is a recently described species of bacterium associated with respiratory disease, growth retardation, mortality, and decreased egg production in chickens and turkeys. Pneumonia, pleuritis, and airsacculitis characterise the infection. ORT has been isolated in many countries but it is still considered exotic in Brazil. Up to date it is prohibited to import and produce reagents for diagnostic and vaccination control. The aim of this study was to isolate and identify the bacteria in chickens. Four isolates were obtained from tracheal swabs of broilers. They were isolated in blood agar with gentamicin and showed biochemical, morphological, antigenic and genetic characteristics of ORT. The results confirm that ORT is present in Brazil.