Draft genome sequence of novel Candidatus Ornithobacterium hominis carrying antimicrobial resistance genes in Egypt.

BACKGROUND: Candidatus Ornithobacterium hominis (O. hominis), which was identified in nasopharyngeal swabs from Egypt, has been associated with respiratory disorders in humans. O. hominis, a recently identified member of the Flavobacteriaceae family, belongs to the largest family within the Bacteroidetes phylum. This family includes hundreds of species and 90 genera, including major human pathogens such as Capnocytophaga canimorsus and Elizabethkingia meningoseptica. Herein, we presented two draft genome assemblies of O. hominis that were extracted from metagenomic data using the Illumina sequencing method. The alignment of reads against the O. hominis genome was accomplished using BLASTN, and the reads with significant hits were extracted using Seqtk and assembled using SPAdes. The primary goal of this study was to obtain a more profound understanding of the genomic landscape of O. hominis, with an emphasis on identifying the associated virulence, antimicrobial genes, and distinct defense mechanisms to shed light on the potential role of O. hominis in human respiratory infections. RESULTS: The genome size was estimated to be 1.84 Mb, including 1,931,660 base pairs (bp), with 1,837 predicted coding regions and a G+C content of 35.62%. Genes encoding gliding motility, antibiotic resistance (20 genes), and the toxA gene were all included in the genome assembly. Gliding motility lipoproteins (GldD, GldJ, GldN, and GldH) and the gliding motility-associated ABC transporter substrate-binding protein, which acts as a crucial virulence mechanism in Flavobacterium species, were identified. The genome contained unique genes encoding proteins, such as the ParE1 toxin that defend against the actions of quinolone and other antibiotics. The cobalt-zinc-cadmium resistance gene encoding the protein CzcB, which is necessary for metal resistance, urease regulation, and colonization, was also detected. Several multidrug resistance genes encoding proteins were identified, such as MexB, MdtK, YheI, and VanC. CONCLUSION: Our study focused on identifying virulence factors, and antimicrobial resistance genes present in the core genome of O. hominis. These findings provide valuable insights into the potential pathogenicity and antibiotic susceptibility of O. hominis.

Ornithobacterium rhinotracheale Isolated from Turkeys over a 20-Year Period Harbor Similar Antimicrobial Susceptibility Profiles and Multidrug Resistance.

Infections with Ornithobacterium rhinotracheale are causing respiratory diseases that require antibiotic treatment in poultry worldwide. In the field, this agent is known to often be resistant to many antimicrobials, complicating therapeutic interventions. Therefore, there is a clear need to monitor trends in resistance development. In the present study, antibiotic resistance profiles of 64 O. rhinotracheale strains isolated from diseased turkeys from 2002 to 2021 were investigated against 19 antimicrobial substances by the microdilution method. Susceptibility toward chloramphenicol, carbapenem, and sulfamethozaxole combination was found for all strains. Most isolates were also susceptible to penicillins (98%-100%), with the exception of oxacillin, cephalosporins (84%-100%), tetracycline (89%), and tylosin (88%). In the case of quinolones, 89% of isolates showed intermediate resistance to enrofloxacin, whereas 90% showed full resistance to nalidixic acid. Full resistance to the tested aminoglycosides and colistin was revealed for all strains. Eighteen different AMR profiles were elucidated; more than half of the isolates (53%) shared the same AMR profile. Similar susceptibility profiles of O. rhinotracheale isolates were found on the different farms, proving some stability over the years. All isolates were classified as multidrug resistant. Multiple outbreaks within a flock or in successive flocks within a farm comprised 46 O. rhinotracheale isolates. Here, occasional changes in susceptibility for some antimicrobial substances were observed. In general, most of the changes occurred in quinolones, followed by tetracycline switching mainly from intermediate resistance to full resistance and vice versa. The present surveillance provides actual data on effective antibiotic treatments in case of disease outbreaks and contributes to the One Health concept acknowledging the important link between animal and human health.

Los Ornithobacterium rhinotracheale aislados de pavos durante un período de 20 años albergan perfiles similares de susceptibilidad a los antimicrobianos y resistencia a múltiples fármacos. Las infecciones por Ornithobacterium rhinotracheale están causando enfermedades respiratorias que requieren tratamiento antibiótico en la avicultura en todo el mundo. En el campo, se sabe que este agente a menudo es resistente a muchos antimicrobianos, lo que complica las intervenciones terapéuticas. Por lo tanto, existe una clara necesidad de monitorear las tendencias en el desarrollo de resistencia. En el presente estudio, se investigaron los perfiles de resistencia a antibióticos de 64 cepas de O. rhinotracheale aisladas de pavos enfermos entre 2002 y 2021 frente a 19 sustancias antimicrobianas mediante el método de microdilución. Se encontró susceptibilidad a la combinación de cloranfenicol, carbapenem y sulfametozaxol para todas las cepas. La mayoría de los aislamientos también fueron susceptibles a las penicilinas (98 %­100 %), con la excepción de oxacilina, cefalosporinas (84 %­100 %), tetraciclina (89 %) y tilosina (88 %). En el caso de las quinolonas, el 89% de los aislados resultaron con susceptibilidad intermedia a la enrofloxacina, mientras que el 90% fueron resistentes al ácido nalidíxico. Todas las cepas revelaron resistencia a los aminoglucósidos y a la colistina probados. Se dilucidaron dieciocho perfiles diferentes de resistencia antimicrobiana; más de la mitad de los aislamientos (53%) compartían el mismo perfil antimicrobiano. Se encontraron perfiles de susceptibilidad similares de aislamientos de O. rhinotracheale en las diferentes granjas, lo que demuestra cierta estabilidad a lo largo de los años. Todos los aislamientos fueron clasificados como resistentes a múltiples fármacos. Los brotes múltiples dentro de una parvada o en parvadas sucesivas dentro de una granja comprendieron 46 aislamientos de O. rhinotracheale. Aquí, se observaron cambios ocasionales en la susceptibilidad a algunas sustancias antimicrobianas. En general, la mayoría de los cambios ocurrieron en las quinolonas, seguido por el cambio de tetraciclina principalmente de resistencia intermedia a resistente y viceversa. La vigilancia actual proporciona datos reales sobre tratamientos antibióticos efectivos en caso de brotes de enfermedades y contribuye al concepto de Una Salud que reconoce el vínculo importante entre la salud humana y animal.

Therapeutic potentials of aivlosin and/or zinc oxide nanoparticles against Mycoplasma gallisepticum and/or Ornithobacterium rhinotracheale with a special reference to the effect of zinc oxide nanoparticles on aivlosin tissue residues: an in vivo approach.

Respiratory diseases inflicted by Mycoplasma gallisepticum (MG) and Ornithobacterium rhinotracheale (ORT) cause severe economic losses and great burden to the poultry industry worldwide. Therefore, the current study was planned to assess the efficacy of aivlosin alone or in combination with zinc oxide nanoparticles (ZnO-NPs) in the treatment of experimental MG and/or ORT infections in broilers. Moreover, we also aimed to evaluate the role of ZnO-NPs on aivlosin residues in broiler tissues. A total of 1,440 Cobb chicks were allocated into 6 groups. At 14 d of age, chickens of groups 1 and 3 were experimentally infected with MG intratracheally and 6 d later, chickens of groups 2 and 3 were infected occulonasaly with ORT. Groups 1, 2, and 3 were divided into 4 subgroups; birds in subgroups 1, 2, and 3 were treated with aivlosin (A), ZnO-NPs (Z), and A/Z, respectively, while those in subgroups 4 was left without treatments. Moreover, groups 4 and 5 were kept noninfected and treated with aivlosin alone or in combination with ZnO-NPs, respectively. Finally, group 6 was kept as a negative control. The current results showed that the recovery from respiratory diseases caused by MG and/or ORT infections was most successful after treatment with A/Z in combination. Consequently, clinical signs, mortality rates, postmortem lesions of the respiratory organs, histopathological lesions of liver, trachea and lung and tracheal MG and ORT counts were significantly (P < 0.05) reduced following A/Z treatment. Taken together, high performance liquid chromatography analysis revealed that ZnO-NPs decreased the aivlosin residues in liver, muscle and skin of healthy and infected chickens. Based on these results, it could be concluded that aivlosin/ZnO-NPs therapy is a valuable approach for controlling MG and/or ORT infections in boilers.

Genetic Diversity of Ornithobacterium rhinotracheale Isolated from Chickens and Turkeys in the United States.

Ornithobacterium rhinotracheale (ORT) is an important bacterial pathogen of great economic significance to poultry production. This bacterium causes severe disease in chickens and turkeys worldwide. The objective of this study was to characterize ORT isolates from two different geographic locations in the United States by multilocus sequence typing (MLST). A total of 60 isolates were included in this study; 36 from California and 24 from Minnesota. All 60 isolates were confirmed to be ORT by PCR that targeted the 16S rRNA gene. The results of MLST revealed eight different sequence types (ST) of ORT. Out of these, four were novel and were assigned numbers ST-32, ST-33, ST-34, and ST-35. ST-1 was the predominant sequence type among all isolates followed by ST-9 and ST-8. Only one isolate was identified as ST-2. No significant variation was seen in STs in ORT isolated from different years. In turkeys, 76.3% (29/38) of isolates belonged to ST-1 and 7.9% (3/38) to ST-8. Of the chicken isolates, 72.2% (13/18) belonged to ST-1 and 16.6% (3/18) to ST-9. Isolates from both states showed low genetic variability. Of the 32 isolates from California, 24 (75%) were identified as ST-1 and 4 (12.5%) were identified as ST-9. The most prevalent sequence type was ST-1 (17/24) followed by ST-8 (3/24) in Minnesota. Three isolates from turkeys in Minnesota belonged to the same ST (ST-8) as the already known ORT strain RefO, which isolated from a rook in Germany in 2000. Whether this sequence type had evolved from wild birds could not be ascertained in this study.

Research Note: Serological investigation of Ornithobacterium rhinotracheale infection in China.

Ornithobacterium rhinotracheale (ORT) has been associated with avian respiratory disease. On coinfection with other pathogens, ORT can cause serious health problems in avian species, leading to financial losses. To monitor the serologic prevalence of ORT in chicken flocks in China, 1,280 sera were collected to determine ORT antibodies among 64 flocks from 15 provinces of China using a commercial ELISA kit. The overall seroprevalence of ORT among the birds tested was 44.06%. In younger chickens, the serum positive rate was lower than that in older chickens, and with increased age, the serum positive rates increased. Older chickens had not only higher positive rates but also higher antibody levels. These data indicated that ORT infections were common in China. Because an ORT vaccine is currently not available, good disease management and biosecurity measures are required for effective disease control.

Genomic Landscape of Ornithobacterium rhinotracheale in Commercial Turkey Production in the United States.

Ornithobacterium rhinotracheale is a causative agent of respiratory tract infections in avian hosts worldwide but is a particular problem for commercial turkey production. Little is known about the ecologic and evolutionary dynamics of O. rhinotracheale, which makes prevention and control of this pathogen a challenge. The purpose of this study was to gain insight into the genetic relationships between O. rhinotracheale populations through comparative genomics of clinical isolates from different U.S. turkey producers. O. rhinotracheale clinical isolates were collected from four major U.S. turkey producers and several independent turkey growers from the upper Midwest and Southeast, and whole-genome sequencing was performed. Genomes were compared phylogenetically using single nucleotide polymorphism (SNP)-based analysis, and then assembly and annotations were performed to identify genes encoding putative virulence factors and antimicrobial resistance determinants. A pangenome approach was also used to establish a core set of genes consistently present in O. rhinotracheale and to highlight differences in gene content between phylogenetic clades. A total of 1,457 nonrecombinant SNPs were identified from 157 O. rhinotracheale genomes, and four distinct phylogenetic clades were identified. Isolates clustered by company on the phylogenetic tree, however, and each company had isolates in multiple clades with similar collection dates, indicating that there are multiple O. rhinotracheale strains circulating within each of the companies examined. Additionally, several antimicrobial resistance proteins, putative virulence factors, and the pOR1 plasmid were associated with particular clades and multilocus sequence types, which may explain why the same strains seem to have persisted in the same turkey operations for decades.IMPORTANCE The whole-genome approach enhances our understanding of evolutionary relationships between clinical Ornithobacterium rhinotracheale isolates from different commercial turkey producers and allows for identification of genes associated with virulence, antimicrobial resistance, or mobile genetic elements that are often excluded using traditional typing methods. Additionally, differentiating O. rhinotracheale isolates at the whole-genome level may provide insight into selection of the most appropriate autogenous vaccine strain, or groups of strains, for a given population of clinical isolates.

Whatman® FTA® Cards Performance for Ornithobacterium rhinotracheale DNA Amplification.

The avian pathogen Ornithobacterium rhinotracheale (ORT) has been implied in the etiology of poultry respiratory disease in recent years. To evaluate whether Whatman® Flinders Technology Associates (FTA®) cards can be used for hazard-free transport and storage of ORT samples for posterior DNA amplification, a controlled assay was performed. Three 10-fold dilutions of an ORT culture suspension were spotted on FTA cards and stored at room temperature (RT) for 6 mo. Sterile swabs were immersed in the same three 10-fold culture dilutions and stored at RT and 4 and -20 C without storage medium for the same time. DNA was extracted from both the FTA cards and swabs 1 day, 1 and 6 wk, and 6 mo following sample preparation and stored at -20 C. At the end of the experiment, real-time PCR amplification of the 16S ribosomal RNA gene was performed from DNA extracted throughout a 6-mo period from all ORT samples stored on both FTA cards and swabs. The obtained threshold cycle values for each ORT DNA extraction date were within the same range for all samples in a dilution-dependent fashion, regardless of storage temperature or used material. Pure ORT colonies could be reisolated 1 day after sample preparation from the swab dilutions stored at all temperatures but not from the FTA cards. We conclude that the efficiency of ORT DNA amplification from samples stored on FTA cards or in swabs is similar. However, FTA cards have the advantage of preventing microorganism growth, thus allowing safe transport and storage, for at least 6 mo, for bacterial dilutions down to at least 104-105 colony-forming units/ml.

A combined retrospective, prospective and experimental study of Ornithobacterium rhinotracheale infection in chickens.

The aim of this study was to examine the histopathological and immunohistochemical changes caused by natural and experimentally-induced Ornithobacterium rhinotracheale infection in the respiratory system of chickens. To this end, three different studies were carried out. The first was a retrospective study of 82 field cases with respiratory disorders compatible with O. rhinotracheale infection. The bacterium was immunohistochemically detected in the lungs in 48 of 82 field cases, and 50 ß-haemolytic (BH) and non-haemolytic (NH) strains were isolated. In the second study, an experimental model of the disease was created using 3-week-old broiler chickens, to identify possible differences of pathogenicity between the BH and NH isolates by the intravenous (IV) and intratracheal (IT) inoculation routes (IR). The group challenged with the NH isolate showed more severe lung lesions than the group challenged with the BH isolate at 7-days postinoculation (p.i.). The 14-day p.i. groups challenged with either the BH or NH isolates by the IT or IV IR had a higher histologic grade of pulmonary and hepatic lesions and a higher total histologic grade of lesions suggesting more severe pathology with longer time of exposure. A direct association between the inoculation routes and the organs affected was shown. Finally, a slaughterhouse study was carried out from October 2014 to May 2015, in which the histologic grade of lesions was significantly higher in immunohistochemically positive for O. rhinotracheale lungs of dead-on-arrival chickens.

Field evaluation of maternal antibody transfer from breeder turkey hens to egg yolks, egg whites, and poults.

Immunoglobulins, which are passed vertically from hens to their progeny, are first present in the eggs but with time also in the developing embryos and eventually in the serum of hatching chicks, and have protective function during embryogenesis and in the first few weeks of birds' life, before the immune system becomes fully efficient. Considering the above fact, the aim of this study was to determine total levels of IgM and IgY as well as specific IgY antibody titers against selected pathogens in the serum of breeder turkeys and their progeny, as well as in egg yolks and egg whites. Study results demonstrated that the level of IgY antibodies in the serum of turkey breeder hens reached 22.04 mg/mL on average in the whole egg laying cycle. In addition, the mean transfer percentage of IgY antibodies from turkey layers to their progeny reached approximately 31.4%, but the level of this transfer differed depending on pathogen character and accounted for 33.2%, 51.9%, 45.1%, and 44.3% in the case of antibodies against avian metapneumoviruses, Newcastle disease virus, Ornithobacterium rhinortacheale, and Pasteurella multocida, respectively. Antibody percentage transfer differed also as affected by the stage of the egg production cycle. Study results confirmed the earlier observed dependency concerning the class of antibodies transferred to eggs from laying hens, and while the IgY were mainly detected in the egg yolk extracts, the IgM were found only in egg white extracts; in comparison to IgY, the IgM antibodies were not transferred to the serum of turkey poults. To our best knowledge, this is the first study that describes in detail the phenomenon of maternal antibody transfer in turkeys.

Method for culturing Candidatus Ornithobacterium hominis.

Candidatus Ornithobacterium hominis has been detected in nasopharyngeal microbiota sequence data from around the world. This report provides the first description of culture conditions for isolating this bacterium. The availability of an easily reproducible culture method is expected to facilitate deeper understanding of the clinical significance of this species.

Phylogenetic relationship of Ornithobacterium rhinotracheale isolated from poultry and diverse avian hosts based on 16S rRNA and rpoB gene analyses.

BACKGROUND: Ornithobacterium (O.) rhinotracheale is an emerging bacterial pathogen in poultry and not fully understood to date. Because of its importance particularly for the global turkey meat industry, reliable diagnostic and characterization methods are needed for early treatment and in future for better vaccine production. The host range of birds infected by O. rhinotracheale or carrying the bacterium in their respiratory tract has constantly increased raising important epidemiological and taxonomic questions for a better understanding of its diversity, ecology and transmission cycles. The purpose of this study was to introduce partial rpoB gene sequencing for O. rhinotracheale into routine diagnostics to differentiate strains isolated from poultry and more diverse avian hosts (i.e., birds of prey, corvids and pigeons) and to compare phylogenetic relationships with results from 16S rRNA gene analysis and multilocus sequence typing (MLST). RESULTS: Partial 16S rRNA gene analysis revealed a high level of homogeneity among the 65 investigated O. rhinotracheale sequences with similarity values ranging from 98.6 to 100% between sequences from non-galliform and poultry species. The corresponding rpoB gene sequences were heterogeneous and ranged in their similarity values from 85.1 to 100%. The structure of the rpoB tree was in strong correlation with previous MLST results revealing three main clusters A (poultry and birds of prey), B (poultry, birds of prey and corvids) and C (pigeons), which were clearly separated from each other. CONCLUSIONS: By using partial sequences from a single gene, the rpoB gene analysis is in good agreement with MLST results with a slight decrease in resolution to distinguish more similar strains. The present results provide strong evidence that traditional phenotypic and genetic methods may not properly represent the heterogeneous group of bacteria classified as O. rhinotracheale. From housekeeping gene analyses, it is very likely that the genus Ornithobacterium includes additional species and partial rpoB gene sequencing can be recommended as fast, cost-effective and readily available method to identify strains and differentiate between O. rhinotracheale and Ornithobacterium-like bacteria.

'Candidatus Ornithobacterium hominis': insights gained from draft genomes obtained from nasopharyngeal swabs.

'Candidatus Ornithobacterium hominis' represents a new member of the Flavobacteriaceae detected in 16S rRNA gene surveys of people from South-East Asia, Africa and Australia. It frequently colonizes the infant nasopharynx at high proportional abundance, and we demonstrate its presence in 42 % of nasopharyngeal swabs from 12-month-old children in the Maela refugee camp in Thailand. The species, a Gram-negative bacillus, has not yet been cultured, but the cells can be identified in mixed samples by fluorescent hybridization. Here, we report seven genomes assembled from metagenomic data, two to improved draft standard. The genomes are approximately 1.9 Mb, sharing 62 % average amino acid identity with the only other member of the genus, the bird pathogen Ornithobacterium rhinotracheale. The draft genomes encode multiple antibiotic-resistance genes, competition factors, Flavobacterium johnsoniae-like gliding motility genes and a homologue of the Pasteurella multocida mitogenic toxin. Intra- and inter-host genome comparison suggests that colonization with this bacterium is both persistent and strain exclusive.

Effect of experimental Ornithobacterium rhinotracheale infection along with live infectious bronchitis vaccination in broiler chickens.

Ornithobacterium rhinotracheale (ORT), a bacterium causing respiratory tract infection, has led to a significant problem in the intensive poultry production in Egypt. Polymerase chain reaction-amplified 784-bp specific ORT DNA fragments were found in 7 ORT isolates from lungs, air sacs, and tracheas of commercial broilers or layers in Egypt in 2015. The objective of this study was to investigate the role of the live variant IBV 4/91 with ORT infection. A total of 120 14-d-old broiler chickens (Cobb 500) were equally divided into 4 groups for experimental infection in a complete randomized design. Group 1 was infected with ORT strain and live infectious bronchitis vaccine (IBV 4/91) simultaneously; group 2 was infected with the bacterial strain alone; group 3 was vaccinated only with IBV 4/91, and group 4 was the non-vaccinated and non-infected control group. The respiratory signs, post-mortem lesions (tracheitis and pneumonia) and histopathological findings of lungs, trachea, and air sacs in the experimentally infected broiler chickens appeared to be more prominent in the chickens of group 1 than group 2. With respect to body weight, weight gain, feed conversion rate, and Ornithobacterium re-isolation, there was a difference (P ≤ 0.05) among the chickens of group 1 and the other groups. This reveals that the use of live infectious bronchitic vaccines, which is a common practice in the local Egyptian field of production, may concomitantly increase the pathogenicity of ORT in broiler chickens.

Serosurvey of Avian metapneumovirus, Orithobacterium rhinotracheale, and Chlamydia psittaci and Their Potential Association with Avian Airsacculitis.

Seasonal outbreaks of airsacculitis in China's poultry cause great economic losses annually. This study tried to unveil the potential role of Avian metapneumovirus (AMPV), Ornithobacterium rhinotracheale (ORT) and Chlamydia psittaci (CPS) in avian airsacculitis. A serological investigation of 673 breeder chickens and a case-controlled study of 430 birds were undertaken. Results showed that infection with AMPV, ORT, and CPS was highly associated with the disease. The correlation between AMPV and CPS were positively robust in both layers and broilers. Finally, we determined the co-infection with AMPV, ORT, and CPS was prevalent in the sampled poultry farms suffering from respiratory diseases and the outbreak of airsacculitis was closely related to simultaneous exposure to all three agents.

Phylogenetic relationship of Ornithobacterium rhinotracheale strains.

The bacterium Ornithobacterium rhinotracheale is associated with respiratory disease in wild birds and poultry. In this study, the phylogenetic analysis of nine reference strains of O. rhinotracheale belonging to serovars A to I, and eight Mexican isolates belonging to serovar A, was performed. The analysis was extended to include sequences from another 23 strains available in the public domain. The analysis showed that the 40 sequences formed six clusters, I to VI. All eight Mexican field isolates were placed in cluster I. One of the reference strains appears to present genetic diversity not previously recognized and was placed in a new genetic cluster. In conclusion, the phylogenetic analysis of O. rhinotracheale strains, based on the 16S rRNA gene, is a suitable tool for epidemiologic studies.

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.

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.

Serosurvey of Avian metapneumovirus, Orithobacterium rhinotracheale, and Chlamydia psittaci and Their Potential Association with Avian Airsacculitis / 生物医学与环境科学（英文）

Seasonal outbreaks of airsacculitis in China's poultry cause great economic losses annually. This study tried to unveil the potential role of Avian metapneumovirus (AMPV), Ornithobacterium rhinotracheale (ORT) and Chlamydia psittaci (CPS) in avian airsacculitis. A serological investigation of 673 breeder chickens and a case-controlled study of 430 birds were undertaken. Results showed that infection with AMPV, ORT, and CPS was highly associated with the disease. The correlation between AMPV and CPS were positively robust in both layers and broilers. Finally, we determined the co-infection with AMPV, ORT, and CPS was prevalent in the sampled poultry farms suffering from respiratory diseases and the outbreak of airsacculitis was closely related to simultaneous exposure to all three agents.

Co-infection of Chlamydia psittaci with H9N2, ORT and Aspergillus fumigatus contributes to severe pneumonia and high mortality in SPF chickens.

Since 2007, most areas of China have seen outbreaks of poultry airsacculitis, which causes hugely economic losses to the poultry industry. However, there are no effective measures to combat the problem. In this study, 105 rations were collected to isolate Aspergillus spp. from the diseased farms. In subsequent experiments, SPF chickens were inoculated with Ornithobacterium rhinotracheale (ORT), Chlamydia psittaci (C. psittaci) and Aspergillus fumigatus (A. fumigatus), and mortality rate, body weight gain and lesion score were evaluated. Of these ration samples, 63 (60.0%) were A. fumigates, 21 (20.0%) were Aspergillus niger (A. niger) and 11 (10.5%) were Aspergillus candidus (A. candidus). Furthermore, SPF birds infected with C. psittaci, ORT, H9N2 virus and A. fumigatus conidia exhibited a mortality rate of 40%, while simultaneous co-infection with C. psittaci, ORT and A. fumigatus resulted in a mortality rate of 20%. The avian airsacculitis was manifested in the C. psittaci + ORT/A. fumigatus, C. psittaci + H9N2 + ORT/A. fumigatus and C. psittaci + H9N2/A. fumigatus groups while others had transient respiratory diseases without mortality. Our survey indicates that feed-borne A. fumigatus is prevalent in poultry rations. The combination of C. psittaci, ORT, H9N2 and A. fumigatus conidia contributes to the replication of avian airsacculitis by aggravating the severe damage to the air sacs and lungs of chickens.

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.