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.

Characterization of Ornithobacterium rhinotracheale from commercial layer chickens in eastern Japan.

From a total of 72 commercial layer and pullet farms that were monitored in the eastern Japan area, 4 farms had mild to severe respiratory disease accompanied by decreased feed intake and drop in egg production. Microbiological analysis showed that 3 of the 4 farms, particularly from Fukushima, Tochigi, and Ibaraki prefectures, were positive for Ornithobacterum rhinotracheale (ORT). Out of 65 birds examined, ORT was isolated in 21 birds (32.31%). All isolates were Gram-negative pleomorphic rods with a colony size of 0.05 mm, translucent with grayish coloration, and with butyric smell after 48 h of incubation in 10% chicken blood agar at 37°C under microaerophilic conditions. All isolates reacted positively in the p-nitrophenyl-ß-d-galactopyranoside test within 3 h and were positive in cytochrome oxidase tests with an API 20NE identification system biocode of 0-0-2-0-0-0-4. An agar gel precipitation test showed that all isolates were serotype-A. All strains were positive in PCR by yielding a 784 bp amplicon of the 16S rRNA gene. All strains were resistant to amikacin, colistin, gentamicin, kanamycin, neomycin, polymyxin b, streptomycin, and sulfamethoxazole trimethoprim and susceptible to amoxicillin clavulanic acid, ampicillin, doxycycline, spectinomycin, and tetracycline. This study is the first characterization of ORT from commercial layer chickens in eastern Japan.

Serotyping, Genotyping, and Antimicrobial Susceptibility of Ornithobacterium rhinotracheale Isolates from Mexico.

The bacterium Ornithobacterium rhinotracheale is associated with respiratory disease and septicemia in poultry. In this study, 9 reference strains and a total of 23 isolates of O. rhinotracheale from respiratory diseased poultry from Mexico were serotyped and genotyped. Furthermore, the antimicrobial susceptibility of isolates and reference strains of O. rhinotracheale were determined. All isolates belong to serotype A and showed a clonal relationship. All reference strains and isolates were resistant to colistin, fosfomycin, gentamicin, kanamycin, streptomycin, and trimethoprim-sulfamethoxazole. These results should eventually be helpful in planning strategies for the control of O. rhinotracheale infections in poultry in Mexico.

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.

Coinfection of Avibacterium paragallinarum and Ornithobacterium rhinotracheale in Chickens from Peru.

The coinfection of Avibacterium paragallinarum and Ornithobacterium rhinotracheale in two outbreaks of infectious coryza from Peru is reported. The diagnosis was confirmed by bacteriologic isolation, PCR testing, and sequencing of the 16S rRNA gene. The susceptibility of the isolates to 12 antimicrobial agents was tested by a disk diffusion method. The isolates were susceptible to amoxicillin/clavulanic acid and florfenicol and were resistant to oxacillin and sulfamethoxazole/trimethoprim. The coinfection of Av. paragallinarum and O. rhinotracheale and the severity of clinical signs were evaluated by experimental infection of specific-pathogen-free chickens. The group inoculated with O. rhinotracheale alone presented minimal clinical signs in 3 of 10 chickens. However, the groups inoculated with both Av. paragallinarum and O. rhinotracheale induced the most-severe clinical signs compared with the group inoculated with Av. paragallinarum alone. In conclusion, coinfections with Av. paragallinarum and O. rhinotracheale may occur, and these outbreaks could be more severe than single infections. Hence, the prevention, control, and diagnosis of Av. paragallinarum with O. rhinotracheale are important in outbreaks of infectious coryza.

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.

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.

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.

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.

Isolation and identification of Ornithobacterium rhinotracheale in broiler breeder flocks of Guilan province, north of Iran.

The aims of the present study were to isolate and serotype, determine the Seroprevalence, Drug susceptibility and diagnosis of infection by Polymerase Chain Reaction (PCR). In this study 460 serum samples and 220 tracheal swabs, 90 ovaries and oviduct swabs, 90 misshapen egg shells swabs were collected from 22 broiler breeder flocks of 5 companies. Serological results showed that all of the 22 flocks (100%) were positive for ORT infection. Ornithobacterium rhinotracheale (ORT) antibodies were detected in 289 (62/83%) out of the 460 serum samples. ORT was detected from tracheal swabs of seven flocks (31/81% or 3/18% out of 220 tracheal swabs). There was significant correlation between flock different ages and ORT titers (p<0.05), but correlation of flock ages and ORT isolates was not significantly different (p>0.05). Seven flocks infected with ORT were detected positive in PCR but bacteria were Isolated from only five culture. No ovaries and oviducts, misshapen egg shell swabs yielded ORT. A 784 bp fragment of the 16S rRNA gene was amplified using ORT specific primers in the PCR. All the isolates were identified as serotype A by Rapid Agglutination Test. Drug sensitivity test using standard disk diffusion technique was performed with 27 antibiotics. Antibiotic susceptibility for Quinolons family was seen more than the others and Cephalosporins family except to Cephalexin. The isolates were 80-100% susceptible to Tetracycline family and the most antibiotic resistant were seen for Aminopenicillins, Polypeptides, Sulfanamides and 80-100% resistant to Aminoglycoside family. Eighty percent of the isolates were resistant to Licomycin and 60% were moderate sensitive to Lincomycin. This study is the first report of prevalence of ORT, bacterial isolation, biochemical characteristics, serotyping and molecular method (PCR) in broiler breeder flocks in Guilan province of Iran.

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.

Characterization of plasmid pOR1 from Ornithobacterium rhinotracheale and construction of a shuttle plasmid.

The bacterium Ornithobacterium rhinotracheale has been recognized as an emerging pathogen in poultry since about 10 years ago. Knowledge of this bacterium and its mechanisms of virulence is still very limited. Here we report the development of a transformation system that enables genetic modification of O. rhinotracheale. The system is based on a cryptic plasmid, pOR1, that was derived from an O. rhinotracheale strain of serotype K. Sequencing indicated that the plasmid consisted of 14,787 nucleotides. Sequence analysis revealed one replication origin and several rep genes that control plasmid replication and copy number, respectively. In addition, pOR1 contains genes with similarity to a heavy-metal-transporting ATPase, a TonB-linked siderophore receptor, and a laccase. Reverse transcription-PCR demonstrated that these genes were transcribed. Other putative open reading frames exhibited similarities with a virulence-associated protein in Actinobacillus actinomycetemcomitans and a number of genes coding for proteins with unknown function. An Escherichia coli-O. rhinotracheale shuttle plasmid (pOREC1) was constructed by cloning the replication origin and rep genes from pOR1 and the cfxA gene from Bacteroides vulgatus, which codes for resistance to the antibiotic cefoxitin, into plasmid pGEM7 by using E. coli as a host. pOREC1 was electroporated into O. rhinotracheale and yielded cefoxitin-resistant transformants. The pOREC1 isolated from these transformants was reintroduced into E. coli, demonstrating that pOREC1 acts as an independent replicon in both E. coli and O. rhinotracheale, fulfilling the criteria for a shuttle plasmid that can be used for transformation, targeted mutagenesis, and the construction of defined attenuated vaccine strains.

In vitro antibiotic resistance profiles of Ornithobacterium rhinotracheale strains from Minnesota turkeys during 1996-2002.

Antimicrobial resistance in nearly all human and animal pathogens is on the increase. In poultry, Ornithobacterium rhinotracheale has been identified as a newly emerging respiratory bacterial pathogen that has caused significant economic losses to the poultry industry. In this study, we examined in vitro antibiotic resistance profiles of 125 isolates of O. rhinotracheale isolated from turkeys in Minnesota during 1996-2002. A majority of isolates was sensitive to clindamycin, erythromycin, spectinomycin, and ampicillin. Resistance against sulfachloropyridiazine decreased from 1996 to 2002, but an increase in resistance was seen against gentamicin, ampicillin, trimethoprim sulfa, and tetracycline. The annual trend slopes for these antibiotics were 7.36%, 3.02%, 2.43%, and 1.95%, respectively. The resistance against penicillin remained constant from year to year with a trend slope of only 0.54% per year. These results emphasize the need for continued monitoring of O. rhinotracheale isolates for antibiotic resistance and establishment of baseline resistance pattern data for this organism. These data can then be used to design and evaluate local epidemiological interventions.