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.

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.

The First Detection of Ornithobacterium rhinotracheale in New Zealand.

Ornithobacterium rhinotracheale (ORT) has been considered exotic to New Zealand and thus, any samples from poultry suspected of ORT infection are submitted as part of an exotic disease investigation managed by Ministry for Primary Industries (MPI) and subjected to standardized test protocols carried out in the physical containment level 3+ laboratory at MPI's Animal Health Laboratory (AHL). All previous exotic disease investigations concerning ORT produced negative results by bacterial culture and conventional PCR. Following the recent introduction of a real-time PCR for ORT at the AHL, several tracheal wash fluids from backyard chickens ( Gallus gallus domesticus ) were tested positive. This identification constituted the first detection of ORT in New Zealand poultry. As a result, a second premise was investigated with further samples testing positive for ORT by molecular assays. This paper describes the two exotic disease investigations associated with the first detection of ORT in New Zealand poultry and its implications.

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.

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.

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.

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.

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.

Significance of interactions between Escherichia coli and respiratory pathogens in layer hen flocks suffering from colibacillosis-associated mortality.

This study aimed to examine the significance of interactions between Escherichia coli and various respiratory pathogens during outbreaks of colibacillosis-associated mortality in layer hen flocks under field conditions. For this purpose, a case-control study involving 20 control flocks with baseline mortality and 20 flocks with increased mortality due to E. coli septicaemia and polyserositis, was conducted. In each colibacillosis flock, blood samples were taken from 20 hens at the onset of clinical disease and three times thereafter at 2-week intervals. Control flocks of comparable ages were sampled in the same way. Pooled sera, taken at the first and last sampling, were examined for antibody titres against infectious bronchitis virus (IBV) and Newcastle disease virus (NDV), and the individual sera from all four samplings were examined for the presence and/or titres of antibodies against avian pneumovirus (APV), Mycoplasma gallisepticum, Mycoplasma synoviae and Ornithobacterium rhinotracheale. Titre increases were seen for IBV D274 (one control flock) and O. rhinotracheale (one control and one colibacillosis flock). An increase in per cent reactors was seen for APV (one control flock), and for M. synoviae (one control and two colibacillosis flocks). The study failed to detect any consistent interactions between E. coli and the aforementioned pathogens. These results indicate that, at least as observed in this study, outbreaks of increased mortality resulting from colibacillosis are not necessarily associated with IBV, NDV, APV, M. gallisepticum, M. synoviae or O. rhinotracheale infections.