Efficacy of ELISA for detection of antibodies against several Ornithobacterium rhinotracheale serotypes

The present investigation were carried out to compare self-made ELISA based on SDS-Antigen extraction of serotype B and one commercial ELISA-kit (Biocheck, Gouda, The Netherlands) for their ability to detect antibodies against 12 ORT serotypes (A-L). Using both ELISA systems, antibodies against all serotypes were detected. Examination of serum samples collected from commercial flocks showed similar results on flock bases. However, some minor variations on sample basis could be demonstrated. In conclusion using both ELISA system antibodies against different serotypes could be detected.     

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.     

In vitro susceptibility of Pseudomonas mallei to antimicrobial agents

Pseudomonas mallei was isolated from pus samples obtained from 34 mallein-positive horses. The isolates were subjected to in vitro sensitivity test using 16 different antimicrobial discs. All isolates (34) were sensitive to sulfamethizole, gentamycin, tetracycline, sulfathiazole, kanamycin, tobramycin, streptomycin and a combination of trimethoprim and sulfamethoxazole while none of them were sensitive to cephalothin, colistin, ampicillin, penicillin and nitrofurantoin. Rifapicin, chloramphenicol and carbenicillin were effective against 32, 26 and 18 isolates respectively. The minimum inhibitory concentrations (MICs) of gentamycin, tetracycline, tobramycin, sulfamethizole, streptomycin, rifampicin and a combination of trimethoprim and sulfamethoxazole were 0.28, 0.38, 0.67, 1.40, 3.40, 5.86 and 5.30 micrograms/ml, respectively.     

In vitro antibiotic sensitivity of strains of Ornithobacterium rhinotracheale isolated in The Netherlands between 1996 and 1999.

All the strains of Ornithobacterium rhinotracheale isolated in the Netherlands between 1996 and 1999 were tested in the agar gel diffusion test for their sensitivity to amoxycillin, tetracycline, enrofloxacin and trimethoprim/sulphonamide (TMP/S). The percentages of strains sensitive to amoxycillin and tetracycline decreased in successive years from approximately 62 per cent to 14 per cent, and four strains were resistant to enrofloxacin or TMP/S. Twelve multiresistant strains were tested against seven alternative antibiotics; they were resistant to all of them except clavulanic acid-potentiated amoxycillin.     

Infection of turkeys with Ornithobacterium rhinotracheale and Mycoplasma synoviae

Within 1 mo, two separate outbreaks of respiratory disease occurred in two flocks on the multiage market turkey farm in Slovenia. More severe dinical signs and higher mortality were observed in male birds. Ornithobacterium rhinotracheale (ORT) was isolated in pure culture from tracheas of the affected birds in both outbreaks. Commercial enzyme-linked immunosorbent assay test showed the presence of antibodies to ORT in sera of birds from both clinically affected flocks and also in two flocks of younger birds without clinical sings. Immunoblotting with ORT culture isolated during the outbreak as an antigen confirmed the presence of antibodies to ORT in sera of turkeys of all four flocks examined. In addition, three different serologic assays also detected antibodies to Mycoplasma synoviae (MS) in three out of four flocks. The concomitant infection with MS did not show an obvious effect on mortality rates nor on the antibody response against ORT. Younger birds appeared to be less susceptible to ORT pathogenicity because in those flocks the infection was subclinical.     

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.     

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.     

In vitro susceptibility of rabbit strains of Clostridium spiroforme to antimicrobial agents

Using an agar dilution method we measured the minimum inhibitory concentration (MIC) of 12 antimicrobial agents against 11 strains of iota-toxigenic strains of Clostridium spiroforme. Each strain was isolated from a separate outbreak of toxic diarrhoea of rabbits. Vancomycin and bacitracin, both agents used to treat intestinal clostridioses of humans and other animals, had a relatively high MIC (8 micrograms/ml or more). Metronidazole was uniformly active against C. spiroforme. With MIC of 8 micrograms/ml or more, both lincomycin (11 strains) and erythromycin (9 strains) were relatively inactive against C. spiroforme, conversely, penicillin G was active (MIC for 8 strains was 0.5 micrograms/ml or less). Exposure to any one of these drugs has been implicated as a predisposing factor for C. spiroforme mediated diarrhoea of rabbits. The greatest variation in MIC was seen for erythromycin (8-fold), penicillin G (8-fold) and tetracycline (16-fold).     

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.     

Use of ovotransferrin as an antimicrobial in turkeys naturally infected with Chlamydia psittaci, avian metapneumovirus and Ornithobacterium rhinotracheale

Respiratory pathogens are difficult to control in large-scale turkey production. This report describes a clinical trial of antimicrobial ovoTF aerosol on a large Belgian turkey farm. ovoTF was administered to reduce Chlamydia psittaci (C. psittaci) infections and to study the impact of this action on the occurrence of Ornithobacterium rhinotracheale (O. rhinotracheale) and avian metapneumovirus (aMPV) infections. Two subsequent broods were included; (i) a control brood receiving no ovoTF and (ii) an ovoTF brood receiving ovoTF aerosol (5mg/animal) at the age of 2 weeks, continuing daily for 12 days. Twenty-four one-day-old toms of the control and ovoTF brood were tagged and monitored for 15 weeks. The control brood experienced two periods of respiratory disease, the first (2-3 weeks of age) due to C. psittaci and the second (8-17 weeks of age) in the presence of C. psittaci, O. rhinotracheale and maybe aMPV. Extensive antibiotic treatment was needed in 2, 8 and 9 week-old toms. In the ovoTF brood, toms stayed healthy until the age of 9 weeks, whereafter respiratory disease occurred in the presence of C. psittaci, O rhinotracheale and aMPV. OvoTF administration: (i) reduced the amount of C. psittaci in the air as demonstrated by bioaerosol monitoring, (ii) prevented respiratory disease during the first half of the brood period, (iii) was associated with 46% reduction of mortality, and (iv) reduced the antibiotic cost. Our results justify additional clinical trials to explore the use of this innovative antimicrobial strategy for poultry.     

Intraspecies genetic variability of Ornithobacterium rhinotracheale in commercial birds in Peru

Strains of the bacterium Ornithobacterium rhinotracheale (ORT), a causal agent of respiratory diseases in birds, were microbiologically isolated, identified, and molecularly characterized. Blood-enriched culture media and biochemistry tests were used for microbiologic identification. Polymerase chain reaction (PCR) and repetitive extragenic palindromic PCR (rep-PCR) techniques were used for molecular identification and characterization, respectively, of the microorganism. ORT strains were isolated in enriched media from the trachea and air sacs of broilers, breeders, and layers from several geographic zones of Peru. Of the original 75 strains isolated from 75 clinical samples from which ORT was recovered during 1998-2000, 25 were selected for further study based on ORT as the primary pathogenic isolate (no other pathogens were detected). Selected isolates were molecularly identified and characterized by PCR using specific primers designed from the conserved zones of the 16S ribosomal genes. Primers used for the identification of ORT produced a specific fragment of 784 base pair (bp), which did not appear in Haemophilus paragallinarum or Pasteurella multocida, microorganisms with similar morphologic and biochemical characteristics that produce dinical signs identical to those of ORT. All 25 strains of ORT tested with rep-PCR had a genetic profile similar to that of ORT American Type Culture Collection 51463, indicating the presence of only one genotype in the ORT strains studied.     

Phenotypic and molecular characterization of isolates of Ornithobacterium rhinotracheale from Peru.

Twenty-five isolates of Ornithobacterium rhinotracheale were examined by agar gel precipitation, immunoperoxidase assay, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, western blot analysis, and a polymerase chain reaction. All of the isolates were identified as serotype A. Protein profiles of whole cell extracts were similar for all the isolates, and a polypeptide with a molecular weight of 33 kD was a major component, being present in all the isolates. In the main, proteins of 33, 42, 52, and 66 kD were recognized in immunoblots with sera from chickens naturally exposed to O. rhinotracheale. A modified polymerase chain reaction assay identified O. rhinotracheale DNA from all the isolates and tracheal swabs, producing amplicons of 784 bp, and distinguished O. rhinotracheale from bacterial agents causing similar clinical signs.     

In vitro antimicrobial susceptibility of anaerobic bacteria isolated from caprine footrot

The in vitro antimicrobial susceptibility of Gram-negative anaerobic bacilli commonly isolated from footrot in goats was studied. A total of 97 isolates belonging to the genera Dichelobacter, Fusobacterium, Prevotella, Porphyromonas and Bacteroides, obtained from clinical cases of footrot in south-western Spain between March 2000 and May 2001, were tested against 25 antimicrobial agents comprising beta-lactams, aminoglycosides, macrolides, chloramphenicol, quinolones, lincosamides, sulphonamides and tetracyclines in order to optimise antibiotic treatment of this disease in goats. beta-lactams, tetracyclines and metronidazole displayed the highest in vitro efficacy against the species involved in the pathogenesis of footrot.     

Isolation and differentiation of a cytochrome oxidase-negative strain of Ornithobacterium rhinotracheale from turkeys

The present study describes the first isolation and identification of a cytochrome oxidase-negative strain of Ornithobacterium rhinotracheale. This strain was isolated from 19-week-old turkeys with severe respiratory signs and was identified using biochemical, cytochemical and serological test methods. These findings indicate that cytochrome oxidase-negative Ornithobacterium rhinotracheale strains become probably also an important rule as a causative agent.     

Outer membrane proteins for serologic detection of Ornithobacterium rhinotracheale infection in turkeys

Ornithobacterium rhinotracheale (ORT) is a bacterium responsible for a respiratory disease in turkeys and chickens and has been identified as one of the emerging respiratory bacterial pathogens. The clinical signs and lesions caused by ORT are very similar to those caused by other respiratory infectious agents; therefore, an accurate diagnostic test is necessary to identify the infection. In this study, we have investigated the use of outer membrane proteins of ORT in an indirect enzyme-linked immunosorbent assay (ELISA) to detect the exposure to ORT infection. Outer membrane proteins of ORT were extracted and used as an antigen in ELISA to detect infection in turkeys exposed to different serotypes of ORT. The ELISA results were compared with the conventional serum plate agglutination test. The agglutination test detected specific antibodies for ORT in 65% of experimentally infected turkeys during the first 2 wk of infection. The ELISA detected up to 100% of infected birds for 8 wk postinfection. The results suggest that ELISA is able to detect the exposure to ORT in later stages of the infection and this assay can be used in serologic surveillance of ORT infection for poultry in the field.     

The susceptibility of isolates of Corynebacterium equi to antimicrobial drugs

Fifty-one isolates of Corynebacterium equi recovered from pigs and horses belonging to two capsular serotypes were tested for susceptibility to antimicrobial agents. No clear differences were detected in sensitivity between isolates of different sources or serotypes. All isolates were sensitive to <0.25 μg/ml of erythromycin and gentamicin. The following minimum inhibitory concentrations (MICs) of antimicrobial agents were determined for ≤90% of isolates: methicillin >16 μg/ml, clindamycin 1–2 μg/ml, tobramycin ≤1 μg/ml, cephalothin 8–64 μg/ml, kanamycin 2–8 μg/ml, amikacin ≤1–2 μg/ml, penicillin 2–≤4 μg/ml, ampicillin 2–8 μg/ml, trimethoprim-sulfa 4/76–32/608 μg/ml tetracycline 1–4 μg/ml and chloramphenicol 8–16 μg/ml.     

Characterisation of some Ornithobacterium rhinotracheale strains and examination of their transmission via eggs

The biochemical characteristics and antibiotic susceptibility of 12 Ornithobacterium rhinotracheale strains isolated from chickens and turkeys suffering from respiratory clinical signs and the survival of some isolates on egg-shell and within chicken eggs during hatching were examined. All O. rhinotracheale strains showed typical biochemical characteristics. Among the 16 drugs examined, penicillin G, ampicillin (MICs ranging from < or = 0.06 microgram/ml to 1 microgram/ml), ceftazidim (with MICs from < or = 0.06 microgram/ml to 0.12 microgram/ml), erythromycin, tylosin, tilmicosin (with some exceptions MICs ranged from < or = 0.06 microgram/ml to 1 microgram/ml) and tiamulin (MICs varied from < or = 0.06 microgram/ml to 2 micrograms/ml) were the most effective. Lincomycin, oxytetracycline and enrofloxacin also gave good inhibitions, but with most strains in a higher concentration (MICs ranged in most cases from 2 micrograms/ml to 8 micrograms/ml). The other antibiotics inhibited the growth of O. rhinotracheale only in very high concentrations (colistin) or not at all (apramycin, spectinomycin, polymyxin B). At 37 degrees C, O. rhinotracheale did not survive on egg-shell for more than 24 hours, while upon inoculation into embryonated chicken eggs it killed embryos by the ninth day, and from the 14th day post-inoculation no O. rhinotracheale could be cultured from the eggs at all. These results suggest that O. rhinotracheale is not transmitted via eggs during hatching.     

Prevalence of antibodies against Ornithobacterium rhinotracheale in broilers and breeders in Southern Brazil

In this investigation, we determined the prevalence of the Ornithobacerium rhinotracheale (ORT) infection in broilers and broiler breeders in southern Brazil. We also correlated the presence of antibodies in broilers with performance. Sera from 1550 broilers from 50 flocks were collected during the slaughter time in nine companies with federal veterinary inspection of the state of Rio Grande do Sul. Sera from 480 meat-type breeders of 40 flocks from 14 companies in southern Brazil were also analyzed by enzyme-linked immunosorbent assay, and the prevalence of antibodies was determined. The prevalence of ORT antibodies in broiler flocks was 63.83%, but in each individual flock only 6.52% of the birds were positive. The prevalence in broiler breeder flocks was 100.00%, and in each individual flock 94.62% of the birds were positive. There was a positive correlation between the presence of antibodies to ORT and decreased body weight in broilers. There was no significant correlation between presence of antibodies to ORT and age, lineage, efficiency index, feed conversion, and mortality. There was a positive correlation between the presence of respiratory signs and antibodies to ORT, although the reverse correlation was not significant. These results confirm that ORT is present and widespread in broilers and broiler breeders in southern Brazil.