Molecular epidemiology of Ornithobacterium rhinotracheale.

Ornithobacterium rhinotracheale is a recently described gram-negative rod-shaped bacterium associated with respiratory tract infections in poultry. In order to determine the molecular epidemiology of this bacterium, we characterized 55 O. rhinotracheale isolates from eight countries on four continents by multilocus enzyme electrophoresis (MLEE), repetitive sequence based-PCR (rep-PCR), and 16S rRNA gene sequencing. MLEE discriminated the O. rhinotracheale isolates into six electrophoretic types (ETs), of which only three ETs were recovered from domesticated poultry. The 16S rRNA gene sequence and rep-PCR analyses confirmed the results obtained by MLEE and indicated limited heterogeneity among isolates of O. rhinotracheale recovered from poultry. Taken together, the results of our analysis demonstrate that the majority of O. rhinotracheale isolates recovered from domesticated poultry throughout the world are represented by a small group of closely related clones and suggest that the bacterium was recently introduced to domesticated poultry from wild bird populations.     

Identification and serotyping of Ornithobacterium rhinotracheale.

In the present study 443 strains of Ornithobacterium rhinotracheale, a causative agent of respiratory disease in fowl, were investigated biochemically and serologically. In both ways O. rhinotracheale could be differentiated from other gram-negative rods and, more particularly, from the Pasteurella-like bacteria potentially pathogenic for fowl. For the biochemical characterization of O. rhinotracheale the API 2ONE identification strip proved to be useful, although O. rhinotracheale is not included in the API system. Serologically, by using monovalent antisera in agar gel precipitation (AGP) tests and enzyme-linked immunosorbent assays (ELISAs), seven serotypes (serotypes A to G) of O. rhinotracheale could be discriminated. The AGP test was chosen as the preferred method to be used for serotyping. Isolates of serotype A were found to be the most prevalent, especially in chickens. Isolates from turkeys were more heterogeneously divided over the serotypes. Some strains showed cross-reactivity between serotypes A, B, and E. Five O. rhinotracheale strains could not be serotyped with the available antisera. Relationships between the geographic origin and the serotypes were found. By the ELISA the presence of antibodies against O. rhinotracheale could be detected in 1-day-old birds as well as in birds with clinical signs, and therefore, it might be useful for diagnostic purposes.     

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.     

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.     

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.     

Synergy between avian pneumovirus and Ornithobacterium rhinotracheale in turkeys

The purpose of this study was to assess the possible synergism between Ornithobacterium rhinotracheale (ORT) and avian pneumovirus (APV), inoculated into turkeys via the natural route, for the reproduction of respiratory disease. Three-week-old specific pathogen free turkeys were inoculated oculonasally with either APV subtype A, ORT or both agents using two different time intervals (3 and 5 days) between APV and ORT. The birds were observed clinically on a daily basis and swabbed intratracheally at short, regular intervals. They were killed at 1, 3, 5, 8 and 15 days post single or dual inoculation and examined for gross lesions at necropsy. Samples of the turbinates, trachea, lungs, air sacs, heart, pericardium and liver were taken for bacteriological and/or histological examination. Combined APV/ORT infections resulted in overt clinical signs and a longer persistence of ORT in the respiratory tract and aggravated the macroscopic and histological lesions in comparison with the groups given single infections. In all ORT-challenged turkeys, ORT was isolated from the turbinates, trachea and lungs, but in turkeys infected with both agents ORT was frequently found in the air sacs and on a single occasion in the heart and pericardium. The time interval between APV and ORT inoculation did not have a significant effect on the outcome of the dual infection. A conspicuous important feature was the attachment of ORT to the cilia of the epithelium of the turbinates and trachea of both ORT-infected and APV/ORT-infected birds. In conclusion, the results show that ORT is able to adhere to and colonize the respiratory tract but, under the circumstances used in this study, is not capable of inducing respiratory disease without viral priming.     

Successful selection of cross-protective vaccine candidates for Ornithobacterium rhinotracheale infection

Ornithobacterium rhinotracheale is a bacterial pathogen known for causing respiratory disease in poultry. In this study, we demonstrate for the first time that cross-protective immunity against different O. rhinotracheale serotypes can be induced by live vaccination. Sera from these live-vaccinated and cross-protected birds were used to identify new vaccine targets by screening an O. rhinotracheale expression library. Out of 20,000 screened plaques, a total of 30 cross-reactive clones were selected for further analysis. Western blot analysis and DNA sequencing identified eight different open reading frames. The genes encoding the eight cross-reactive antigens were amplified, cloned in an expression vector, and expressed in Escherichia coli. Purified recombinant proteins with a molecular mass ranging from 35.9 kDa to 62.9 kDa were mixed and tested as a subunit vaccine for (cross-)protection against challenge with homologous and heterologous O. rhinotracheale serotypes in chickens. Subunit vaccination resulted in the production of antibodies reactive to the recombinant proteins on Western blot, and this eight-valent vaccine conferred both homologous and heterologous protection against O. rhinotracheale challenge in chickens.     

Immunohistochemical and serological investigation of experimental Ornithobacterium rhinotracheale infection in chickens

Immunohistochemical techniques were used to prove that Ornithobacterium rhinotracheale was the causative agent of lesions in the air sacs and lungs in chickens, but only after infection with Newcastle Disease virus (NDV). At first, the bacteria attached to the epithelium of the air sacs. Subsequently, they infiltrated the air sacs, and caused thickening of the air sacs, the formation of oedematous and granulomatous tissue, and accumulation of macrophages. The infection peaked at 5 to 9 days, after which recovery was seen. In the lungs, some areas with bronchially-associated lymphoid tissue were affected. The other organs investigated were shown not to be affected. In the absence of NDV infection, aerosol exposure of chickens to O. rhinotracheale only resulted in minimal and temporary microscopic air sac lesions. No O. rhinotracheale cells or fragments could be detected at any time point later than 2 days post-exposure. In spite of the absence of visible lesions, chickens exposed to O. rhinotracheale without prior NDV infection reacted serologically. The duration and the titre of this immune response was indistinguishable from that obtained in chickens exposed after NDV infection. Thus, infection with O. rhinotracheale appears to be restricted to the respiratory tract, with lesions only evident in birds previously infected with NDV, even though a strong serological response can be established in the absence of prior viral infection.     

The effect of vaccination of pullets against Ornithobacterium rhinotracheale infection.

The effect of vaccination of chickens with different inactivated vaccines against experimental Ornithobacterium rhinotracheale challenge was investigated. Eight different vaccines, with different inactivating substances (Formalin and thiomersal) and with or without adjuvant (mineral oil, alum and aluminium hydroxide gel), were produced. Following vaccination of experimental chickens at week 8 with formalin-inactivated mineral oil adjuvant bacterin, the mean O. rhinotracheale antibody titres rose to 5.88 2log 21 days after primary vaccination and enhanced to a titre of 6.59 2log 21 days after booster vaccination. The bacterin in mineral oil adjuvant induced the highest serologic response and a significant decrease of lesions such as air sacculitis and pneumonia in vaccinated birds compared with the unvaccinated challenge control birds. The bacterin in either alum or aluminium hydroxide gel adjuvant induced a moderate serologic response and a decrease of lesions compared with the unvaccinated challenge controls. The study showed that vaccination of layer chicken at the eighth week followed by a booster dose at the 12th week of age can effectively protect against O. rhinotracheale infections.     

Antibiotic sensitivity and resistance in Ornithobacterium rhinotracheale strains from Belgian broiler chickens

Establishing the antibiotic sensitivity of the avian respiratory pathogen Ornithobacterium rhinotracheale is difficult because of the organism's complex growth requirements and the unusually frequent occurrence of resistance. The minimal inhibitory concentrations of 10 antibiotics were determined for 45 strains of O. rhinotracheale from Belgian broiler chickens collected from 45 farms between 1995 and 1998. They were compared with the type strain, which was isolated from a turkey, and a strain isolated from a rook. All the broiler strains were resistant to lincomycin and to the β -lactams ampicillin and ceftiofur. Less than 10% of the strains were sensitive to the macrolides tylosin and spiramycin, tilmicosin and flumequine. A few strains were sensitive to enrofloxacin and doxycycline. All strains were sensitive to tiamulin.     

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.     

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 10⁸ 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.     

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.     

In vivo selection of reduced enrofloxacin susceptibility in Ornithobacterium rhinotracheale and its resistance-related mutations in gyrA

This study determines the genetic background of the change in antimicrobial susceptibility to enrofloxacin of Ornithobacterium rhinotracheale (ORT) isolates with increased MIC values, isolated either from the field or from turkeys treated with enrofloxacin under experimental challenge conditions. In the field strains of ORT that were either less susceptible or, occasionally, resistant to enrofloxacin, point mutations had occurred in amino acids at positions 83 (serine) or 87 (aspartic acid) of the GyrA subunit. In the isolates showing reduced susceptibility following experimental enrofloxacin treatment (increase in MIC from < or =0.03 to 0.25 microg/ml), molecular analysis revealed a constantly recurring point mutation (G-->T) at nucleic acid position 646 (E. coli numbering) of gyrA resulting in an amino acid change from aspartic acid to tyrosine at position 87 of the GyrA subunit, which is a known hot spot for fluoroquinolone resistance. This study indicates that a single course of enrofloxacin treatment may contribute to the selection of the first mutant with reduced fluoroquinolone susceptibility in ORT. Acquired fluoroquinolone resistance is commonly encountered in ORT isolates. This is the first time that the causal mechanism of fluoroquinolone resistance in ORT has been investigated.     

The effect of Ornithobacterium rhinotracheale vaccination of broiler breeder chickens on the performance of their progeny

The effect of Ornithobacterium rhinotracheale vaccination of broiler breeders on antibody titres and performance of breeders and broilers was investigated. O. rhinotracheale antibody titres and performance data were recorded from 16 different broiler breeder flocks and from 79 of their broiler progeny flocks. Eight breeder flocks were vaccinated with an inactivated O. rhinotracheale vaccine while the other eight breeder flocks were left unvaccinated against this bacterium. Following vaccination, mean O. rhinotracheale antibody titres in the breeders rose to a 6.5 log 2 units higher value than in unvaccinated breeders, and remained at a mean titre of 15 log 2 units during the entire production period. This resulted in significantly higher maternal antibody titres against O. rhinotracheale in the broiler progeny of vaccinated breeder flocks compared with the offspring of unvaccinated flocks. Statistical analyses revealed no differences in performance between vaccinated and unvaccinated breeders. There was a significantly lower mean mortality rate and higher mean production index in the broilers derived from vaccinated breeders.     

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.     

Serologic prevalence of Ornithobacterium rhinotracheale infections in broilers and broiler breeder chickens in Kermanshah Province,west of Iran

The presence of Ornithobacterium rhinotracheale (ORT) in poultry farms in several parts of Iran has been confirmed over the last few years. The purpose of this study was to determine the presence of ORT antibodies by enzyme-linked immunosorbent assay (ELISA) in broiler and broiler breeder chickens in western parts of Iran. In a one-year period, from April 2009 to February 2010, a total of 435 blood samples were collected from 30 commercial chicken flocks (24 broiler flocks and six broiler breeder flocks) located in Kermanshah Province, west of Iran. None of the broilers and broiler breeders have been vaccinated against ORT prior to sampling. The province was divided into four geographical areas: southwest, southeast, northwest, and northeast. Flocks in each area, specifically the birds in each flock, were randomly sampled. The presence of antibodies against ORT in each serum sample was tested twice by ELISA using a commercial kit. The results revealed that 50.1 % broiler and 70.5 % broiler breeder chickens were serologically positive for ORT. Out of 347 serum samples obtained from broiler chickens, 174 (50.1 %) were positive for ORT antibodies, which represented 18 (75.0 %) of 24 examined broiler flocks. A higher rate of seropositivity (71.4 % of samples and 83.3 % of broiler flocks) was observed in the northwest. Out of 88 samples obtained from broiler breeder chickens, 62 (70.5 %) were positive for ORT antibodies, which belonged to six (100 %) of the examined broiler breeder flocks. Detection of anti-ORT antibodies among broiler breeders was significantly higher than that of broilers.     

A comparative study on detection of Ornithobacterium rhinotracheale antibodies in meat-type turkeys by dot immunobinding assay,rapid agglutination test and serum agglutination test

The objectives of the present study were to develop a dot-immunobinding assay (DIA) and a serum agglutination test (SAT) for detection of Ornithobacterium rhinotracheale , to compare the rapid agglutination test (RAT) and the SAT, and to make a serosurvey of O. rhinotracheale exposure on turkey farms in Turkey. Antiserum against O. rhinotracheale bacterin was prepared in rabbits and 72 serum samples were collected from turkeys with respiratory signs on four farms. Comparison of the tests showed that 55.5, 48.6 and 40.3% of serum samples were positive by RAT, SAT and DIA, respectively. The sensitivity of the DIA appeared to be lower than that of the agglutination tests but the specificity is not known.     

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.     

Comparison of the efficacy of four antimicrobial treatment schemes against experimental Ornithobacterium rhinotracheale infection in turkey poults pre-infected with avian pneumovirus.

The clinical efficacy of drinking-water administration of enrofloxacin for 3 and 5 days, amoxicillin for 5 days and florfenicol for 5 days for the treatment of respiratory disease induced by an experimental Ornithobacterium rhinotracheale infection in turkeys pre-infected with avian pneumovirus (APV) was assessed based on clinical, bacteriological and histopathological examinations. Experimental groups of 15 susceptible 3-week-old turkeys were each inoculated oculonasally with APV subtype A and 3 days later with susceptible O. rhinotracheale bacteria. Antimicrobial treatment started 1 day after O. rhinotracheale inoculation. After infection, the birds were examined and scored for clinical signs, swabbed daily and weighed at different times. Five birds were euthanized and examined for macroscopic lesions at necropsy at 5 days post bacterial inoculation, and the remainder at 15 days post bacterial inoculation. Samples of the turbinates, trachea, lungs, air sacs, heart and pericardium were collected for bacteriological and/or histological examination. Recovery from respiratory disease caused by an APV/O. rhinotracheale dual infection was most successful after enrofloxacin treatment, irrespective of treatment duration, followed by florfenicol. Amoxicillin treatment was not efficacious. Clinical signs and the number of O. rhinotracheale organisms re-isolated from the trachea and the different respiratory organs were significantly reduced by enrofloxacin treatment for 3 and 5 days. O. rhinotracheale bacteria were not re-isolated from the tracheas of the birds treated with enrofloxacin except for one bird in the 5-day group, as early as 1 day after medication onset. In the group treated with enrofloxacin for 5 days, O. rhinotracheale organisms with a higher minimal inhibitory concentration value (x8) were isolated starting 2 days following treatment onset, initially from a single turkey and subsequently from the other animals.