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.     

Correlation between ability of Ornithobacterium rhinotracheale to agglutinate red blood cells and susceptibility to fosfomycin

Twenty five freeze-dried isolates of Ornithobacterium rhinotracheale were used for the determination of minimum inhibitory concentrations (MIC) against the antibiotic fosfomycin (Fosbac, produced by Bedson SA, consisting of a 25% mixture of fosfomycin). The same isolates were tested for their ability to haemagglutinate chicken red blood cells. Ten of the 25 isolates were found to be susceptible to fosfomycin (MIC values below 128 ug/ml). All of these isolates were able to agglutinate red blood cells. This is the first report on the ability of O. rhinotracheale to agglutinate red blood cells. The remaining 15 isolates were resistant to fosfomycin (MIC values above 128 ug/ml). Only five of these isolates were found to have the ability to agglutinate red blood cells. There appears to be a correlation between the ability of O. rhinotracheale isolates to agglutinate red blood cells and their susceptibility to fosfomycin. The ability of certain isolates of O. rhinotracheale to agglutinate red blood cells, raises the questions of differences in virulence between the isolates which can agglutinate red blood cells and those which cannot and the use of this ability to agglutinate red blood cells as an alternative method for serotyping O. rhinotracheale.     

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.     

鼻气管鸟杆菌对SPF雏鸡的致病性

通过气管、消化道和肌肉注射接种鼻气管鸟杆菌（Ornithobacterium rhinotracheale,ORT）人工感染SPF雏鸡,观察试验鸡的发病情况及症状,并检测各试验组和对照组的体质量;利用建立的PCR检测方法对试验组和对照组的排菌情况进行动态观察;于感染后不同时间随机剖杀试验组和对照组鸡只,采集心脏、肝脏、肺脏和气管等组织固定、切片、H.E.染色,观察各器官病理组织学变化,利用建立的IFA方法对ORT在雏鸡体内的分布进行研究,并进行血常规和血液生化指标检测。结果表明,ORT接种后,主要引起雏鸡的呼吸困难、咳嗽等症状,增重明显降低;病理组织学变化以肝脏、心脏组织细胞变性,气管和肺脏广泛性出血和炎性细胞浸润为特征;IFA阳性信号主要分布于肺脏和气管;从感染后2～8d一直可持续排菌;HGB、WBC、RBC、BUN和AST发生显著变化。这表明ORT感染主要引起雏鸡的呼吸系统病变,气管接种比消化道和肌肉注射接种对雏鸡致病性强,肺脏和气管是ORT侵害的主要靶器官。     

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.     

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.     

Survival of Ornithobacterium rhinotracheale in sterilized poultry litter

Ornithobacterium rhinotracheale (ORT) has been associated with respiratory disease, increased mortality, retarded growth, and decreased egg production in chickens and turkeys. Surveillance of exposure to ORT infection in the field has shown that prevalence of the infection is higher during winter months. The ability of ORT to remain viable in the poultry litter was studied at different temperatures over time. Presterilized poultry litter was inoculated with 10(11) colony-forming units of ORT and kept at -12 C, 4 C, 22 C, 37 C, and 42 C. Reisolation and titration of ORT from litter was attempted at intervals. Results indicate that ORT survived for 1 day at 37 C, 6 days at 22 C, 40 days at 4 C, and at least 150 days at -12 C. ORT did not survive 24 hr at 42 C. The survival of ORT at lower temperatures may be associated with the higher incidence of ORT infection in poultry during winter months.     

Identification and characterization of Ornithobacterium rhinotracheale isolates from Mexico

Ten gram-negative, pleomorphic, rod-shaped isolates from coryza-like, respiratory diseased laying and broiler chickens were identified as Ornithobacterium rhinotracheale. All O. rhinotracheale isolates showed typical biochemical and enzymatic characteristics. Also, all isolates showed hemagglutinating activity with glutaraldehyde-fixed erythrocytes. On the basis of this property, a rabbit-raised antiserum was produced for an isolate. All isolates were identified by antiserum by hemagglutination-inhibition tests. No cross-reactions were observed when O. rhinotracheale isolates were tested with Haemophilus paragallinarum antisera, and vice versa. Mild respiratory signs, including mild nasal discharge, slight rales, and sneezing, were observed in challenged chickens. At postmortem examination, multifocal pneumonia, airsacculitis, and foamy exudate in abdominal cavity were observed. Furthermore, because bacterial adherence is regarded as an essential step in the infection process, in vitro adherence of O. rhinotracheale isolates to chicken tracheal epithelial cells was tested. All isolates showed positive adherence. Obtained results indicate that O. rhinotracheale is a pathogenic agent present in the Mexican poultry.     

Adherence of five serovars of Ornithobacterium rhinotracheale to chicken tracheal epithelial cells

1. Interaction between bacteria and host tissue is important, both for primary adhesion and tissue-specific colonisation, as well as for pathogen invasion for different host tissues.

2. Ornithobacterium rhinotracheale is a bacterium associated with respiratory tract infections in poultry. The mechanisms by which O. rhinotracheale causes infection are not known. To date, at least 18 serovars of this bacterium, with or without the ability to agglutinate erythrocytes of chicken and other species, have been identified.

3. The purpose of this work was to evaluate the ability of five references strains, belonging to serovars A, B, C, D and E, to adhere to a culture of primary chicken tracheal cells.

4. Serovars A and B adhered to less than 20% of tracheal cells with no specific adherence pattern. Serovars C, D and E gave adherence values greater than 70%. Serovars C and E showed a diffuse adherence pattern, while serovar D had an aggregated adherence pattern.

5. The adherence ability and pattern could be associated with different pathogenicity mechanisms in the various serovars but more studies are needed to understand the reasons for these differences.     

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.     

In vitro susceptibility of field isolates of Francisella tularensis subsp. holarctica recovered in Spain to several antimicrobial agents

Forty-two recent (1997-1999) Spanish isolates of Francisella tularensis subsp.holarctica were tested in a broth microdilution method for their susceptibilities to 29 antimicrobial agents, including penicillins, cephalosporins, cephamicins, monobactams, penems, aminoglycosides, tetracyclines, macrolides, quinolones, chloramphenicol and fosfomycin. The isolates were resistant to beta-lactam antibiotics and susceptible to chloramphenicol, ciprofloxacin, levofloxacin and norfloxacin.     

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.     

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.     

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).     

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.     

猪肺炎支原体体外抗菌药物敏感性试验研究

为了解猪肺炎支原体对抗菌药物的敏感性,采用宏量肉汤稀释法对4株猪肺炎支原体的最小抑菌浓度（MIC）值进行了测定。结果表明,猪肺炎支原体对泰妙菌素和环丙沙星最为敏感,MIC≤0．03μg／mL,其次分别为四环素类药物（包括四环素和多西环素）、林可霉素和泰乐菌素,而对氟苯尼考的敏感性则较差。本研究可为猪支原体肺炎的防控以及抗菌药物的合理使用提供参考。