Draft genome sequence of novel Candidatus Ornithobacterium hominis carrying antimicrobial resistance genes in Egypt.

BACKGROUND: Candidatus Ornithobacterium hominis (O. hominis), which was identified in nasopharyngeal swabs from Egypt, has been associated with respiratory disorders in humans. O. hominis, a recently identified member of the Flavobacteriaceae family, belongs to the largest family within the Bacteroidetes phylum. This family includes hundreds of species and 90 genera, including major human pathogens such as Capnocytophaga canimorsus and Elizabethkingia meningoseptica. Herein, we presented two draft genome assemblies of O. hominis that were extracted from metagenomic data using the Illumina sequencing method. The alignment of reads against the O. hominis genome was accomplished using BLASTN, and the reads with significant hits were extracted using Seqtk and assembled using SPAdes. The primary goal of this study was to obtain a more profound understanding of the genomic landscape of O. hominis, with an emphasis on identifying the associated virulence, antimicrobial genes, and distinct defense mechanisms to shed light on the potential role of O. hominis in human respiratory infections. RESULTS: The genome size was estimated to be 1.84 Mb, including 1,931,660 base pairs (bp), with 1,837 predicted coding regions and a G+C content of 35.62%. Genes encoding gliding motility, antibiotic resistance (20 genes), and the toxA gene were all included in the genome assembly. Gliding motility lipoproteins (GldD, GldJ, GldN, and GldH) and the gliding motility-associated ABC transporter substrate-binding protein, which acts as a crucial virulence mechanism in Flavobacterium species, were identified. The genome contained unique genes encoding proteins, such as the ParE1 toxin that defend against the actions of quinolone and other antibiotics. The cobalt-zinc-cadmium resistance gene encoding the protein CzcB, which is necessary for metal resistance, urease regulation, and colonization, was also detected. Several multidrug resistance genes encoding proteins were identified, such as MexB, MdtK, YheI, and VanC. CONCLUSION: Our study focused on identifying virulence factors, and antimicrobial resistance genes present in the core genome of O. hominis. These findings provide valuable insights into the potential pathogenicity and antibiotic susceptibility of O. hominis.

Case reports involving coinfection with Avibacterium paragallinarum and Ornithobacterium rhinotracheale in broiler chickens and Avibacterium endocarditis in broiler breeding hens in Poland.

ABSTRACTThe study describes three clinical cases of infection with Avibacterium spp.. In case no. 1, respiratory clinical signs and high mortality (0.7-4.2% daily; total 21.2%) in Ross 308 broiler chickens were shown to be caused by coinfection with sequence type 9 of O. rhinotracheale presumptive serotype A and A. paragallinarum presumptive serotype B. The identical (pulsed-field gel electrophoresis) restriction pattern (pulsotype) of seven A. paragallinarum isolates indicated that infectious coryza in broilers was caused by the same clone. In cases 2 and 3, sudden increased deaths in Ross 308 broiler breeders (especially males) with lesions in the endocardium (valvular or mural endocarditis) were shown to be caused by A. endocarditis. Among nine antibiotics tested, florfenicol was the only antibiotic to which all A. paragallinarum and O. rhinotracheale isolates were susceptible. Out of the eight antibiotics tested, 11 A. endocarditis isolates from both clinical cases of infective endocarditis were susceptible to penicillin, amoxicillin, doxycycline and florfenicol. The A. endocarditis isolates tested in both clinical cases had different PFGE patterns (pulsotypes), but identical within a case. The causes of infectious coryza and infective endocarditis in the cases presented have not been determined. In the prevention of infectious diseases in large-scale livestock farming, it is very important to follow the rules of biosecurity.

Genomic Landscape of Ornithobacterium rhinotracheale in Commercial Turkey Production in the United States.

Ornithobacterium rhinotracheale is a causative agent of respiratory tract infections in avian hosts worldwide but is a particular problem for commercial turkey production. Little is known about the ecologic and evolutionary dynamics of O. rhinotracheale, which makes prevention and control of this pathogen a challenge. The purpose of this study was to gain insight into the genetic relationships between O. rhinotracheale populations through comparative genomics of clinical isolates from different U.S. turkey producers. O. rhinotracheale clinical isolates were collected from four major U.S. turkey producers and several independent turkey growers from the upper Midwest and Southeast, and whole-genome sequencing was performed. Genomes were compared phylogenetically using single nucleotide polymorphism (SNP)-based analysis, and then assembly and annotations were performed to identify genes encoding putative virulence factors and antimicrobial resistance determinants. A pangenome approach was also used to establish a core set of genes consistently present in O. rhinotracheale and to highlight differences in gene content between phylogenetic clades. A total of 1,457 nonrecombinant SNPs were identified from 157 O. rhinotracheale genomes, and four distinct phylogenetic clades were identified. Isolates clustered by company on the phylogenetic tree, however, and each company had isolates in multiple clades with similar collection dates, indicating that there are multiple O. rhinotracheale strains circulating within each of the companies examined. Additionally, several antimicrobial resistance proteins, putative virulence factors, and the pOR1 plasmid were associated with particular clades and multilocus sequence types, which may explain why the same strains seem to have persisted in the same turkey operations for decades.IMPORTANCE The whole-genome approach enhances our understanding of evolutionary relationships between clinical Ornithobacterium rhinotracheale isolates from different commercial turkey producers and allows for identification of genes associated with virulence, antimicrobial resistance, or mobile genetic elements that are often excluded using traditional typing methods. Additionally, differentiating O. rhinotracheale isolates at the whole-genome level may provide insight into selection of the most appropriate autogenous vaccine strain, or groups of strains, for a given population of clinical isolates.

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.

A combined retrospective, prospective and experimental study of Ornithobacterium rhinotracheale infection in chickens.

The aim of this study was to examine the histopathological and immunohistochemical changes caused by natural and experimentally-induced Ornithobacterium rhinotracheale infection in the respiratory system of chickens. To this end, three different studies were carried out. The first was a retrospective study of 82 field cases with respiratory disorders compatible with O. rhinotracheale infection. The bacterium was immunohistochemically detected in the lungs in 48 of 82 field cases, and 50 ß-haemolytic (BH) and non-haemolytic (NH) strains were isolated. In the second study, an experimental model of the disease was created using 3-week-old broiler chickens, to identify possible differences of pathogenicity between the BH and NH isolates by the intravenous (IV) and intratracheal (IT) inoculation routes (IR). The group challenged with the NH isolate showed more severe lung lesions than the group challenged with the BH isolate at 7-days postinoculation (p.i.). The 14-day p.i. groups challenged with either the BH or NH isolates by the IT or IV IR had a higher histologic grade of pulmonary and hepatic lesions and a higher total histologic grade of lesions suggesting more severe pathology with longer time of exposure. A direct association between the inoculation routes and the organs affected was shown. Finally, a slaughterhouse study was carried out from October 2014 to May 2015, in which the histologic grade of lesions was significantly higher in immunohistochemically positive for O. rhinotracheale lungs of dead-on-arrival chickens.

Adherence of Ornithobacterium rhinotracheale to chicken embryo lung cells as a pathogenic mechanism.

Ornithobacterium rhinotracheale is a bacterium that causes respiratory disease in birds and it has been isolated in countries with a large poultry production, including Mexico. The pathogenicity mechanisms of this bacterium have not been completely elucidated yet. The capacity of the bacterium to adhere to epithelial cells of chicken in vitro has been evidenced, and since this bacterium has been isolated from the lungs and air sacs of several avian species, the aim of this study was to determine if this bacterium can adhere to chicken lung cells. We used five O. rhinotracheale reference serovars (A-E) that were in contact with primary lung cells cultured from a 19-day-old chicken embryo. O. rhinotracheale adherence was evaluated through optical and transmission electron microscopies. The results revealed that O. rhinotracheale is capable of adhering to chicken embryo lung cells within 3 h of incubation with a diffuse adherence pattern. The adherence percentages of the chicken embryo lung cells were 51-96% according to the serovar of the bacterium. Relative adherence was from 4 to 8 bacteria per cell. Transmission electron microscope data revealed intracellular bacteria inside a vacuole in less than 3 h of incubation.

Serosurvey of Avian metapneumovirus, Orithobacterium rhinotracheale, and Chlamydia psittaci and Their Potential Association with Avian Airsacculitis.

Seasonal outbreaks of airsacculitis in China's poultry cause great economic losses annually. This study tried to unveil the potential role of Avian metapneumovirus (AMPV), Ornithobacterium rhinotracheale (ORT) and Chlamydia psittaci (CPS) in avian airsacculitis. A serological investigation of 673 breeder chickens and a case-controlled study of 430 birds were undertaken. Results showed that infection with AMPV, ORT, and CPS was highly associated with the disease. The correlation between AMPV and CPS were positively robust in both layers and broilers. Finally, we determined the co-infection with AMPV, ORT, and CPS was prevalent in the sampled poultry farms suffering from respiratory diseases and the outbreak of airsacculitis was closely related to simultaneous exposure to all three agents.

Characterization of Ornithobacterium rhinotracheale field isolates from Hungary.

Ornithobacterium rhinotracheale is a widely distributed rod-shaped Gram-negative bacterium that infects several avian species including chickens and turkeys. It is associated with respiratory signs, growth retardation, mortality, and reduced egg production, thus causing severe economic losses to the poultry industries. In this study, 37 field isolates of O. rhinotracheale, collected from various locations in Hungary between 1997 and 2015, were identified and characterized by the analysis of partial 16S rRNA gene sequences, enterobacterial repetitive intergenic consensus (ERIC)-PCR, and random amplified polymorphic DNA (RAPD) PCR assays with the OPG11, OPH19, and M13 primers. Most of the field isolates were serotype A, one was serotype B, and four were serotype D. One isolate could not be typed with antisera against serotypes A-E. In a phylogenetic analysis of the 16S rRNA sequences, the isolates formed two clusters. Thirteen distinct patterns were identified with ERIC-PCR, and the RAPD assay with the M13 primer assigned the isolates to 10 different patterns. The other two RAPD assays were unsuitable for distinguishing and grouping the isolates. Neither ERIC type nor RAPD pattern correlated with the place or year of isolation. However, the strains isolated from chickens were more heterogeneous on ERIC-PCR than the isolates recovered from turkeys. In this study, ERIC-PCR was the most discriminatory method for investigating the genetic diversity of O. rhinotracheale isolates.

Comparative pathomorphological, bacteriological and serological examination of broiler breeders and pheasants experimentally infected with Ornithobacterium rhinotracheale.

The aim of the investigations was to determine the influence of Ornithobacterium rhinotracheale (ORT) on the development of pathomorphological lesions in the respiratory organs and on the health status of experimentally infected broiler breeders and pheasants from the rearing stage. There was no evidence of clinical signs in infected broiler breeder hens nor in the group of infected pheasants except for one bird in the latter group which exhibited slower movement and gasping. The frequency and intensity of pathomorphological lesions were higher in pheasants. The gross pathology findings were characterized mainly by redness of the mucosa of the upper respiratory tract and accumulation of mucous content in the nasal cavities, infraorbital sinuses, larynx and trachea. Histopathology confirmed the presence of inflammation of the upper respiratory tract. Lesions in the lungs included hyperaemia, granulomatous and fibrinous pneumonia. ORT was reisolated only from the group of infected pheasants. Reisolation was successful from the respiratory organs (trachea, larynx, infraorbital sinuses, and lungs) of eight out of 10 infected birds. The serological response in both species was characterized by rapid production of specific antibodies that reached a maximum level in the blood in the first week after experimental infection. The antibody titres decreased gradually and were maintained at a stable level until the 12th week after inoculation. Fourteen weeks post-inoculation specific antibodies could not be detected by enzyme-linked immunosorbent assay.

Efficacy of gamithromycin against Ornithobacterium rhinotracheale in turkey poults pre-infected with avian metapneumovirus.

Ornithobacterium rhinotracheale is an avian respiratory pathogen that affects turkeys. The objective of this study was to evaluate the clinical efficacy of gamithromycin (GAM) against O. rhinotracheale in turkeys. The birds were inoculated oculonasally with 10(8) colony-forming units (cfu) of O. rhinotracheale, preceded by infection with avian metapneumovirus. In addition to a negative (CONTR-) and a positive control group (CONTR+) there were two treated groups administered GAM (6 mg/kg) either subcutaneously (GAM SC) or orally (GAM PO) by administration as a single bolus at one-day post-bacterial infection (p.b.i.). From the start of the avian metapneumovirus infection until the end of the experiment, the turkeys were examined clinically and scored daily. In addition, tracheal swabs were collected at several days p.b.i. Necropsy was performed at 4, 8 and 12 days p.b.i. to evaluate the presence of gross lesions, and to collect trachea and lung tissue samples and air sac swabs for O. rhinotracheale quantification. The clinical score of the GAM SC group showed slightly lower values and birds recovered earlier than those in the GAM PO and CONTR+ groups. O. rhinotracheale cfus were significantly reduced in tracheal swabs of the SC group between 2 and 4 days p.b.i. At necropsy, CONTR+ showed higher O. rhinotracheale cfu in lung tissues compared to the treated groups. Moreover, at 8 days p.b.i. only the lung samples of CONTR+ were positive. In conclusion, the efficacy of GAM against O. rhinotracheale was demonstrated, especially in the lung tissue. However, the PO bolus administration of the commercially available product was not as efficacious as the SC bolus.

Conditions that induce biofilm production by Ornithobacterium rhinotracheale.

Ornithobacterium rhinotracheale (ORT) is a Gram-negative bacillus that causes respiratory disease in birds, and directly affects the poultry industry. The mechanisms behind these infections are not completely known. Currently, its capacity to form biofilms on inert surfaces has been reported; however, the conditions for biofilm development have not been described yet. The present work was aimed at identifying the conditions that enhance in vitro biofilm formation and development by ORT. For this, serovars A-E were analysed to assess their ability to induce biofilm development on 96-well flat-bottom polystyrene microtitre plates under diverse conditions: temperature, incubation time, and CO2 concentration. The results obtained showed not only that all serovars have the ability to produce in vitro biofilms, but also that the optimal conditions for biofilm density were 40°C after 72 h at an elevated CO2 concentration. In conclusion, ORT biofilm formation depends on the environmental conditions and may contribute to the persistence of this microorganism.

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.

Pharmacokinetic and pharmacodynamic properties of gamithromycin in turkey poults with respect to Ornithobacterium rhinotracheale.

The macrolide gamithromycin (GAM) has the ability to accumulate in tissues of the respiratory tract. Consequently, GAM might be a suitable antibiotic to treat bacterial respiratory infections in poultry, such as Ornithobacterium rhinotracheale. As O. rhinotracheale infections are common in turkey flocks, the aim of this study was to determine the pharmacokinetic (PK) parameters of GAM in plasma, lung tissue, and pulmonary epithelial lining fluid (PELF) of turkeys and to correlate them with pharmacodynamic (PD) characteristics (PK/PD). The animal experiment was performed with 64 turkeys, which received either a subcutaneous (SC, n=32) or an oral (PO, n=32) bolus of 6 mg GAM/kg body weight (BW). GAM concentrations in plasma, lung tissue, and PELF were measured at different time points post administration (p.a.), and PK characteristics were determined using non-compartmental modeling. The maximum plasma concentration after PO administration was ten-fold lower than after SC injection (0.087 and 0.89 µg/mL, respectively), whereas there was no difference in lung concentrations between both routes of administration. However, lung concentrations at day 1 p.a. were significantly higher than plasma levels for both routes of administration (2.22 and 3.66 µg/g for PO and SC, respectively). Consequently, lung/plasma ratios were high, up to 50 and 80 after PO and SC administration, respectively. GAM could not be detected in PELF, although this might be attributed to the collection method of PELF in birds. The GAM minimum inhibitory concentration (MIC) was determined for 38 O. rhinotracheale strains; MIC50 and MIC90 were 2 and >32 µg/mL, respectively. PK/PD correlation for lung tissue demonstrated that the time above the MIC90 of the susceptible population (2 µg/mL) was 1 day after PO bolus and 3.5 days after SC administration. The area under the curve (AUClast)/MIC ratios for lung tissue after SC and PO administration were 233 and 90, respectively. To conclude, GAM is highly distributed to lung tissue in turkey poults, suggesting that it has the potential to be used to treat respiratory infections such as O. rhinotracheale.

Biochemical and immunological responses of young turkeys to vaccination against Ornithobacterium rhinotraheale and different levels of dietary methionine.

The objective of this study was to verify the hypothesis that increasing levels of dietary methionine can stimulate the mechanisms of cell-mediated and humoral immunity in young turkeys. The blood and organs involved in cell-mediated and humoral immune responses were analyzed in 8-week-old turkeys that had been vaccinated against Ornithobacterium rhinotraheale (ORT) infection (on days 17 and 48). The birds were fed diets with a low (LM), medium (MM) and high (HM) methionine content (0.45 and 0.40%, 0.60 and 0.51%, 0.71 and 0.57% in weeks 1 - 4 and 5 - 8, respectively). Dietary methionine supplementation led to a significant increase in body weights of turkeys at 56 days of age, from 3532 g in group LM to 3720 g in group MM and 3760 g in group HM (p=0.001). A significant increase in vaccine-induced antibody titers against ORT was noted in group HM relative to group LM (p=0.006). Increasing levels of methionine had no significant effect on total serum IgG nor IgM levels and most serum biochemical parameters, TP, ALB, GLOB, GLU, AST, ALP, P and Ca. In comparison with group LM, group HM turkeys were characterized by a lower percentage of IgM⁺ B cell subpopulation in the blood and bursa of Fabricius. The percentages of CD4⁺ and CD8⁺ T cell subpopulations in the bursa of Fabricius in group HM were significantly different from those found in groups LM and MM. The highest percentages of CD4⁺ T cells and CD8⁺ T cells in the spleen were observed in groups LM (p<0.001) and HM (p=0.04), respectively. The differences were statistically significant relative to the remaining groups. Turkeys of group LM were characterized by a lower CD4⁺ T cell percentage in the thymus (p<0.001) and a lower CD8⁺ T cell percentage in the cecal tonsils (CTs) (p<0.01). Vaccination against ORT resulted in a significant increase in the percentage of CD4⁺CD8⁺ T cell subpopulation and a decrease in the percentage of CD8⁺ T cell subset in the spleen.

Experimental comparison of hemolytic and nonhemolytic Ornithobacterium rhinotracheale field isolates in vivo.

Ornithobacterium rhinotracheale (ORT) is a nonhemolytic, gram-negative, pleomorphic, rod-shaped bacterium that causes upper and lower respiratory tract disease in poultry. Recently, hemolytic strains of ORT have been isolated with increasing frequency from field outbreaks. A study was conducted to determine whether the hemolytic phenotype is associated with any change in virulence. Briefly, 225 turkey poults, vaccinated against hemorrhagic enteritis at 4 wk of age, were randomly divided into nine replicates housed in separate rooms: three sham treatment controls (25 poults/replicate), three challenged with a nonhemolytic (NH) field isolate (24 poults/replicate), and three challenged with a hemolytic (H) field isolate (24 poults/replicate). Nine days postvaccination, poults were inoculated intratracheally with either 0.2 ml sterile phosphate-buffered saline (PBS), 2 x 10(8) colony-forming units (CFU) of the NH isolate in 0.2 ml PBS, or 2 x 10(8) CFU of the H isolate in 0.2 ml PBS. Serum and body weights were obtained at 0, 7, 14, and 21 days postinoculation (dpi). Tissues were taken for culture and histopathology from five randomly selected poults/replicates at 7, 14, and 21 dpi. When compared with poults inoculated with the H isolate or controls, those inoculated with the NH isolate showed a highly significant depression in weight gain at 7 dpi. NH poults also had significantly higher levels of antibody against ORT at 14 and 21 dpi. Reisolations decreased over time and, by 21 dpi, only the NH phenotype could be found. Based on a Likert-type scale, poults inoculated with the NH isolate had significantly higher histopathologic lesion scores in lung tissue at 7, 14, and 21 dpi. Results suggest that nonhemolytic field isolates are more virulent then hemolytic ones. These findings are unusual because hemolytic phenotypes are often more virulent in other bacterial species.

Isolation, identification, antibiotic resistance profile and molecular analysis of Ornithobacterium rhinotracheal isolates from turkeys.

BACKGROUND: Ornithobacterium rhinotracheal (ORT) infects numerous birds, particularly chickens and turkeys. ORT is an emerging bacterial pathogen of global concern in the poultry industry. As ORT is rapidly spreading throughout commercial poultry, it requires intensive studies of its epidemiology, diagnostic procedures, molecular typing, virulence genes and antimicrobial resistance. OBJECTIVES: The present study was conducted in isolation and identification of ORT from slaughtered turkeys. METHODS: Cleft palate swabs of 200 were collected from slaughtered turkeys and cultured on blood agar. ORT was characterized using biochemical tests and PCR targeting the ORT 16S rRNA gene. Virulence genes of isolates were determined targeting adenylate kinase (adk), copA and virulence-associated protein D (vapD) genes. Additionally, diversity of ORT isolates was performed by enterobacterial repetitive intergenic consensus (ERIC) and RAPD PCR. Disk diffusion was used to determine the antibiotic sensitivity of the isolates. RESULTS: ORT was identified in 23 (11.5%) samples using both the biochemical tests and PCR. The result of detecting virulence genes showed that all the isolates (23: 100%) had the adk gene, whereas two (8.7%) isolates had the copA gene, and seven (30.43%) isolates had the vapD gene. Molecular typing of isolates revealed 21 different patterns by RAPD PCR assay using M13 primer and 20 distinct patterns by ERIC PCR test. Both ERIC and RAPD PCR were distinctive methods for investigating the genetic diversity of ORT isolates. The antibiotic resistance test showed that 18 (78.26%) isolates were resistant to gentamicin, amikacin, cefazolin, streptomycin and penicillin. All isolates (100%) were resistant to cloxacillin and fosfomycin. CONCLUSIONS: This study showed the prevalence of ORT in turkey and high resistance of this bacterium to many common veterinary antibiotics. Moreover, both ERIC and RAPD PCR are distinctive methods for investigating the genetic diversity of ORT isolates. These data may help monitor antibiotic resistance and typing of ORT in epidemiological studies and serve as the foundation for designing region-specific vaccines for future use.

Isolation and characterization of small-colony variants of Ornithobacterium rhinotracheale.

Ornithobacterium rhinotracheale is a Gram-negative bacterium associated with respiratory diseases in many avian species, with worldwide distribution, and it causes significant economic loss to the poultry industry. In this study, the isolation and characterization of O. rhinotracheale small-colony variants (SCVs) are described for the first time. O. rhinotracheale isolates (n = 27) were recovered from tracheal samples (n = 321) collected from different avian species with clinical signs of respiratory disease. Of the 27 O. rhinotracheale isolates, 21 (77.8%) showed SCVs in their primary cultures. Five O. rhinotracheale SCV isolates showed high levels of stability and were chosen for further characterization with their wild-type (WT) isolates. Stable O. rhinotracheale SCVs were oxidase negative, while their WT isolates were positive. Growth curves for stable O. rhinotracheale SCVs indicated lower growth rates and longer lag phases than for their WT isolates. Furthermore, it was possible to increase the efficacy of the broth medium in supporting the growth of O. rhinotracheale WT isolates by supplementing it with 5% fetal bovine serum (FBS) and 2% IsoVitaleX Enrichment. Antibiotic susceptibility tests showed that O. rhinotracheale SCVs had higher MIC values than their WT isolates. This study suggests that successful antibiotic treatment of respiratory diseases associated with O. rhinotracheale must take into consideration the resistance patterns of O. rhinotracheale SCVs. Intracellular persistence in murine RAW 264.7 macrophages revealed that O. rhinotracheale SCV28 had higher survival rates than its WT isolate. Finally, small-colony variants may be important contributors to the pathogenesis of O. rhinotracheale.

Clinical efficacy of florfenicol administered in the drinking water against Ornithobacterium rhinotracheale in turkeys housed in different environmental conditions: a pharmacokinetic/pharmacodynamic approach.

In poultry rearing, medicated drinking water is a commonly used administration route, but drug uptake can be affected by many factors. In this study, the influence of two important parameters, the photoperiod and feeding schemes, on florfenicol uptake in turkeys was tested. First, the uptake was determined as the pharmacokinetic/pharmacodynamic profile of florfenicol; and second, we evaluated the clinical efficacy of florfenicol against Ornithobacterium rhinotracheale. Both experiments were conducted during a 5-day treatment of 30 mg/kg body weight florfenicol administered via drinking water and considering different photoperiods and feeding schemes (group 20/4L: photoperiod of 20 h, fed ad libitum; group 16/8L: photoperiod of 16 h, fed ad libitum; group 16/8R: photoperiod of 16 h, fed ad libitum but feed was withdrawn during the dark period and replaced 1 h after lighting). On day 1 of treatment, all groups showed plasma concentrations above the minimum inhibitory concentration (both MIC50 and MIC90, 1 mg/l) of 37.7%, 63.5% and 53.1% of a 24-h interval for 20/4L, 16/8L and 16/8R, respectively. Only in the 16/8L and 16/8R groups was the MIC also exceeded on day 5 (47.9% and 21.5% of a 24-h interval, respectively). In all groups, a clinical improvement could be noticed, resulting in reduction of the clinical score. However, only the 16/8L and 16/8R groups showed significant differences from the control group. The results demonstrated an important influence of the photoperiod on the pharmacokinetics of florfenicol as well as the clinical outcome in an infection model. It can be advised that the photoperiod should be <20 h to have sufficient drug intake. Nevertheless, there was no effect between fed and fasted turkeys for both the pharmacokinetics and the clinical outcome.

Demonstration of Ornithobacterium rhinotracheale in pheasants (Phasianus colchicus) with pneumonia and airsacculitis.

Outbreaks of respiratory disease were investigated in reared pheasants (Phasianus colchicus) aged approximately 18 to 32 weeks, released into the semi-wild on four shooting estates in southern England. The clinical signs in the affected birds included swelling of the face and eyes, loss of condition, gasping respirations and coughing. The gross pathology findings included sinusitis, airsacculitis, pleural oedema and lung lesions. The histopathological findings in the affected lungs were characterized by a granulomatous pneumonia. Ornithobacterium rhinotracheale (ORT) was isolated from respiratory tract tissues, and 16S rRNA gene sequencing on three isolates revealed two distinct genotypes, one previously associated with some electrophoretic type (ET) 1 strains and the other a novel genotype that clustered among sequences previously associated with ET 3, ET 4, ET 5 and ET 6 isolates. The localization of ORT within the lung tissue was demonstrated by fluorescent in-situ hybridization in the bronchial exudate of three cases, although not within the granulomatous lesions themselves. In each case, ORT was identified as part of a complex of other respiratory agents including avian paramyxovirus type 2, avian coronavirus, Mycoplasma gallisepticum, Mycoplasma synoviae and other Mycoplasma species, Escherichia coli, Pasteurella multocida, other Pasteurellaceae and Syngamus trachea, suggesting synergism with other agents. Exposure to other intercurrent factors, including adverse weather conditions and internal parasitism, may also have exacerbated the severity of disease.

Development of real-time polymerase chain reaction assay for detection of Ornithobacterium rhinotracheale in poultry.

Ornithobacterium rhinotracheale (ORT) is an emerging bacterium causing severe economic losses in poultry mostly due to respiratory and locomotory disturbances. Due to the fastidious nature of the organism, ORT is often overgrown by faster-growing commensal and pathogenic bacteria. In this study we developed a real-time polymerase chain reaction (qPCR) assay for rapid and sensitive detection of ORT in samples collected from chickens and turkeys. The qPCR assay developed was able to detect 17 reference strains of ORT (serotypes A to Q) tested in this study, and no false-positive results were obtained from other organisms associated with respiratory tract infections. The qPCR assay was 100 times more sensitive than the modified conventional PCR. Using tenfold serial dilutions of the recombinant plasmid DNA containing the target gene fragment, the detection limit of the qPCR was estimated to be > or = 100 plasmid copies per reaction. Out of 42 examined poultry flocks, 26 cases were tested positive by both assays. The qPCR assay reduces turnaround time to about 2 hr, two times faster than the modified conventional PCR.