Ornithobacterium Rhinotracheale Research Articles

A detailed analysis of 16S rRNA gene sequencing and conventional PCR-based testing for the diagnosis of bacterial pathogens and discovery of novel bacteria.

This study represents the first analysis of the bacterial community in chickens affected by swollen head syndrome, utilizing 16S rRNA gene sequencing. Samples were obtained from clinical laying chickens and were examined for the presence of Avibacterium paragallinarum (APG) and Ornithobacterium rhinotracheale (ORT) using conventional polymerase chain reaction (PCR). From the samples, five APG-positive (APG) and APG-negative (N-APG) samples were chosen, along with five specific pathogen-free chickens, for 16S rRNA gene sequencing. Results showed that APG and ORT were widely detected in the chicken samples with swollen head syndrome (SHS, 9/10), while APG was detected in all five specific pathogen-free (SPF) samples. In contrast, conventional PCR sensitivity was found to be inadequate for diagnosis, with only 35.7% (5/14) and 11.1% (1/9) sensitivity for APG and ORT, respectively, based on 16S rRNA gene sequencing data. Furthermore, 16S rRNA gene sequencing was able to quantify the bacteria in the samples, revealing that the relative abundance of APG in the APG group ranged from 2.7 to 81.3%, while the relative abundance of APG in the N-APG group ranged from 0.1 to 21.0%. Notably, a low level of APG was also detected in all 5 SPF samples. The study also identified a significant number of animal and human common bacterial pathogens, including but not limited to Gallibacterium anatis, Riemerella columbina, Enterococcus cecorum, Mycoplasma synoviae, Helicobacter hepaticus, and Staphylococcus lentus. In conclusion, 16S rRNA gene sequencing is a valuable tool for bacterial pathogen diagnosis and the discovery of novel bacterial pathogens, while conventional PCR is not reliable for diagnosis.

Concurrent infection of Infectious Bronchitis Virus and Mycoplasma gallisepticum in a backyard poultry

This study reports the co-existence of two S1 mutants of GI-13 (4/91-like) Infectious Bronchitis Virus (IBV) and Mycoplasma gallisepticum (MG) in a backyard poultry flock that had non-vaccinated 30 broiler chickens and four turkey pullets. Serum samples and tracheal swabs were taken from the chickens and turkey pullets showing respiratory signs. Serum antibody levels were measured using commercial ELISA kits against IBV, Avian Influenza Virus (AIV), Newcastle Disease Virus (NDV), Avian Metapneumovirus (AMPV), MG, Mycoplasma synoviae (MS), and Ornithobacterium rhinotracheale (ORT). Additionally, tracheal swabs were tested for AIV serotypes H5, H7, and H9, NDV, IBV, AMPV, MG, MS, Pasteurella multocida, Avibacterium paragallinarum, and Bordetella avium by circular amplification technology (CAT). Anti-MS, -IBV, -MG, -NDV, -AMPV, and -ORT IgG antibodies were detected in some chicken sera, while anti-NDV, -MG, -MS, and -ORT IgG antibodies were detected in turkey sera. All avian tracheal swabs were positive for MG. However, IBV was only detected in chicken tracheal samples tested by CAT. The IBV strains were genotyped by sequencing a part of the S1 glycoprotein gene. The alignment analyses of two isolates showed 99.35% and 98.69% nucleotide similarities and 99.02% amino acid similarities with the 4/91 IBV vaccine and field strains. Two mutants showed 99.35% nucleotide and 100% amino acid sequence identity to each other. The turkeys and chickens in the flock had MG and MG/IBV co-infections, respectively. Consequently, the presence of mutants of 4/91 (GI-13) IBV genotypes and MG found in backyard poultry could be a potential epidemiological source for commercial flocks in poultry integrations.

Case reports involving coinfection with Avibacterium paragallinarum and Ornithobacterium rhinotracheale in broiler chickens and Avibacterium endocarditis in broiler breeding hens in Poland

ABSTRACT The study describes three clinical cases of infection with Avibacterium spp.. In case no. 1, respiratory clinical signs and high mortality (0.7–4.2% daily; total 21.2%) in Ross 308 broiler chickens were shown to be caused by coinfection with sequence type 9 of O. rhinotracheale presumptive serotype A and A. paragallinarum presumptive serotype B. The identical (pulsed-field gel electrophoresis) restriction pattern (pulsotype) of seven A. paragallinarum isolates indicated that infectious coryza in broilers was caused by the same clone. In cases 2 and 3, sudden increased deaths in Ross 308 broiler breeders (especially males) with lesions in the endocardium (valvular or mural endocarditis) were shown to be caused by A. endocarditis. Among nine antibiotics tested, florfenicol was the only antibiotic to which all A. paragallinarum and O. rhinotracheale isolates were susceptible. Out of the eight antibiotics tested, 11 A. endocarditis isolates from both clinical cases of infective endocarditis were susceptible to penicillin, amoxicillin, doxycycline and florfenicol. The A. endocarditis isolates tested in both clinical cases had different PFGE patterns (pulsotypes), but identical within a case. The causes of infectious coryza and infective endocarditis in the cases presented have not been determined. In the prevention of infectious diseases in large-scale livestock farming, it is very important to follow the rules of biosecurity.

Unraveling frontiers in poultry health (part 1) – Mitigating economically important viral and bacterial diseases in commercial Chicken and Turkey production

This symposium offered up-to-date perspectives on field experiences and the latest research on significant viral and bacterial diseases affecting poultry. A highlight was the discussion on the use of enteroids as advanced in vitro models for exploring disease pathogenesis. Outcomes of this symposium included identifying the urgent need to improve the prevention and control of avian influenza by focusing research on vaccine effectiveness. In this regard, efforts should focus on enhancing the relatedness of vaccine antigen to the field (challenge) virus strain and improving immunogenicity. It was also revealed that gangrenous dermatitis could be controlled through withholding or restricting the administration of ionophores during broiler life cycle, and that administration of microscopic polymer beads (gel) based-live coccidia vaccines to chicks could be used to reduce necrotic enteritis-induced mortality. It was emphasized that effective diagnosis of re-emerging Turkey diseases (such as blackhead, fowl cholera, and coccidiosis) and emerging Turkey diseases such as reoviral hepatitis, reoviral arthritis, Ornithobacterium rhinotracheale infection, and strepticemia require complementarity between investigative research approaches and production Veterinarian field approaches. Lastly, it was determined that the development of a variety of functionally-specific enteroids would expedite the delineation of enteric pathogen mechanisms and the identification of novel vaccine adjuvants.

Prospects for the creation of an oral vaccine against ornithobacteriosis of birders

The work was carried out at the pharmacogenomics laboratory of the Institute of Chemical Biology and Fundamental Medicine of the Siberian Branch of the Russian Academy of Sciences and in the molecular biology sector of the Siberian Federal Scientific Center for Agrobiotechnologies of the Russian Academy of Sciences. Ornithobacterium rhinotracheale DNA was provided by poultry farms of the Republic of Mordovia of the Russian Federation. In affected birds, inflammatory changes were observed in the larynx, upper part of the trachea and infraorbital sinuses, and fibrin clots were observed in the respiratory sacs. Pathological material was subcultured in brain heart broth in the presence of antibiotics. Analysis of subcellular localisation of the OR77 protein was carried out using the SignalP-6.0 method. The immune epitope database (IEDB) was used to search for B-dependent epitopes of the OR77 gene. Primers for cloning were designed, and cloning regions of the OR77 gene containing B-dependent epitopes were simulated by ligating BamHI and HindIII into the polylinker of the shuttle vector pBE-S. Experiments were carried out to clone the active epitope sequence of the O. rhinotracheale OR77 protein in Bacillus subtilis and use it orally to identify immune activity and other possible adverse reactions. Nucleotide sequence analysis was performed using the UniproUGENEv program. The amplification procedure was carried out. The prospect of the possible synthesis of a new, inexpensive, and effective vaccine against the pathogen O. rhinotracheale is described.

Mycoplasma gallisepticum and Mycoplasma synoviae in Turkeys in Poland.

The pathogenic mycoplasmas are among the bacteria causing significant losses in the poultry industry worldwide. Mycoplasma gallisepticum (MG) and M. synoviae (MS) are economically important pathogens causing chronic respiratory disease, decreased growth, egg production and hatchability rates, and significant downgrading of carcasses. Effective diagnosis of infection with these species in poultry is highly requisite considering their two routes of spreading-horizontal and vertical. Their prevalence and molecular epidemiology were investigated in 184 turkey flocks in Poland. Tracheal samples were selected from 144 broiler flocks and 40 turkey breeder flocks collected in 2015-2023. The prevalence of MG was determined by real-time PCR targeting the 16S rRNA gene and PCR targeting the mgc2 gene, and MS was determined by a 16-23S rRNA real-time PCR and a vlhA gene PCR. Further identification and molecular characterization were carried out using PCR and sequencing. M. gallisepticum and M. synoviae were found in 8.33% and 9.72% of turkey broiler flocks respectively. The phylogenetic analysis of MG isolates in most cases showed high similarity to the ts-11-like strains. MS isolates showed high similarity to strains isolated from flocks of laying hens causing EAA. Additional tests detected Ornithobacterium rhinotracheale, Gallibacterium anatis, Enterococcus faecalis and Enterococcus faecium, Staphylococcus aureus and Riemerella anatipestifer. These secondary pathogens could have significantly heightened the pathogenicity of the mycoplasma infections studied.

Development and Validation of PCR Diagnostic Assays for Detection of Avibacterium paragallinarum and Ornithobacterium rhinotracheale.

PCR is the most effective method for detecting difficult-to-cultivate pathogens and pathogens that are part of mixed infections in animals, such as Ornithobacterium rhinotracheale, which causes bird ornithobacteriosis, or Avibacterium paragallinarum, which causes infectious coryza. In this work, we developed and validated two efficient and sensitive diagnostic assays for the rapid and accurate detection of A. paragallinarum and O. rhinotracheale DNA in bacterial isolates and clinical samples using real-time PCR with TaqMan-like probes. When designing the PCR assays, we performed in silico analysis, optimized DNA isolation methods and PCR conditions, and assessed the analytical and diagnostic performance of PCR. We designed primers and probes that have no mismatches with published whole-genome sequences of bacteria. The optimization of conditions showed that the PCR assays are sufficiently robust to changes in temperature and oligonucleotide concentration. The validation showed that the developed assays have high analytical and diagnostic sensitivity and specificity. These assays are expected to improve the differential diagnosis of respiratory diseases in chickens and turkeys.

Evaluasi Efektivitas Antibiotik Komersil Terhadap Agen Penyebab Gejala Snot pada Ayam Broiler di Kabupaten Kupang

Snot is a symptom of upper respiratory system infections in poultry, characterized by exudate production from the nasal cavity, swelling of infraorbital sinuses, snoring, sneezing, and dyspnoea. The aetiology of snot that have been isolated are Avibaterium paragallinarum, Pasteurella multocida, Ornithobacterium rhinotracheale, and Mycoplasma sp. Snot symptom can be eradicated with antibiotic treatment; however, antibiotic resistance makes antibiotic treatment ineffective. This study is aimed to evaluate the effectiveness of some commercial antibiotics against bacteria isolated from broiler chicken with snot symptom in Kupang Regency. Amoxycillin, ampicillin, tetracycline, cefoxitin, and ciprofloxacin were tested using the Kirby Bauer method with McFarland Turbidity Standard against Avibacterium paragallinarum, Ornithobacterium rhinotracheale, Pasteurella multocida, and Mycoplasma sp isolates. Inhibition zones were measured and compared to the standard of the Clinical Laboratory Standard Institute (CLSI) to determine the sensitivity or resistance percentage. The result showed that Avibacterium paragallinarum, Ornithobacterium rhinotracheale, Pasteurella multocida, and Mycoplasma sp were highly resistant to amoxycillin and ampicillin, yet most sensitif to ciprofloxacin. This suggests commercial antibiotics that are productive to eradicate snot symptom and implies some antibiotics that are ineffective to overcome snot symptom and hence should not be used in the field.

Molecular Detection of The Ornithobacterium rhinotracheale From Turkeys Flocks in Alexandria Governorate During 2020 – 2021

Molecular Detection of The Ornithobacterium rhinotracheale From Turkeys Flocks in Alexandria Governorate During 2020 – 2021

Description and genome-wide analysis of Profundicola chukchiensis gen. nov., sp. nov., marine bacteria isolated from bottom sediments of the Chukchi Sea.

Two Gram-negative, aerobic halophilic non-motile strains designated KMM 9713 and KMM 9724T were isolated from the bottom sediments sampled from the Chukchi Sea in the Arctic Ocean, Russia. The novel strains grew in 0.5-5% NaCl, at 7-42°C, and pH 5.5-10.5. Phylogenetic analyses based on 16S rRNA gene and whole genome sequences revealed that strains KMM 9713 and KMM 9724T were close to each other and shared the highest 16S rRNA gene sequence similarity of 91.28% with the type strain Ornithobacterium rhinotracheale DSM 15997T and 90.15-90.92% with the members of the genus Empedobacter in the family Weeksellaceae. Phylogenetic trees indicated that strains KMM 9713 and KMM 9724T formed a distinct line adjacent to their relative O. rhinotracheale DSM 15997T. The average nucleotide identity values between strain KMM 9724T and O. rhinotracheale DSM 15997T, Empedobacter brevis NBRC 14943T, and Moheibacter sediminis CGMCC 1.12708T were 76.73%, 75.78%, and 74.65%, respectively. The novel strains contained the predominant menaquinone MK-6 and the major fatty acids of iso-C17:0 3-OH, iso-C15:0 followed by iso-C17:1ω6. Polar lipids consisted of phosphatidylethanolamine, one an unidentified aminophospholipid, two unidentified aminolipids, and two or three unidentified lipids. The DNA G+C contents of 34.5% and 34.7% were calculated from genome sequence of the strains KMM 9713 and KMM 9724T, respectively. Based on the phylogenetic evidence and distinctive phenotypic characteristics, strains KMM 9713 and KMM 9724T are proposed to be classified as a novel genus and species Profundicola chukchiensis gen. nov., sp. nov. The type strain of Profundicola chukchiensis gen. nov., sp. nov. is strain KMM 9724T (= KACC 22806T).

Detection of Ornithobacterium rhinotracheale antibodies in broiler by ELISA in Mosul city

Detection of Ornithobacterium rhinotracheale antibodies in broiler by ELISA in Mosul city

The effect of early administration of antibiotics or feeding a diet containing a coccidiostat on inflammatory responses and the morphological structure of selected organs of the immune system in young meat-type turkeys

It was assumed that early administration of enrofloxacin or doxycycline may impair immune function and alter the morphology of organs of the immune system in turkeys, and that diets containing the coccidiostat monensin, an ionophore antibiotic, can exert similar effects. The aim of this study was to determine whether early antibiotic administration or feeding a diet containing a coccidiostat affect immune function in young turkeys. The experiment had a completely randomized design, with 8 groups (a total of 3,080 one-day-old turkeys), 7 replicate pens per group and 55 birds per pen. The experiment had a 2-factorial design, with 4 treatments (C-control, M-monensin, E-enrofloxacin, and D-doxycycline) and 2 groups of birds (vaccinated and unvaccinated) per treatment. Control group birds did not receive the coccidiostat or antibiotics. Group M was administered monensin at 90 mg/kg feed for the first 5 d of life, group E received enrofloxacin at 10 mg/kg BW, added to drinking water, for the first 5 d of life, and group D received doxycycline at 50 mg/kg BW, added to drinking water, for the first 5 d of life. One-day old turkeys from groups C+, M+, E+, and D+ were administered live-attenuated vaccines against turkey rhinotracheitis (TRT) (Poulvac TRT; Zoetis, Parsippany, NJ) and Newcastle disease (ND) (Nobilis ND clone 30; Merck, Rahway, NJ) by coarse spray; 28-day-old birds were administered a subcutaneously injected inactivated vaccine against Ornithobacterium rhinotracheale (ORT) (Ornitin, Phibro, Poland). Turkeys from groups C-, M-, E-, and D- were not vaccinated. It was found that early administration of enrofloxacin or doxycycline, or feeding a diet containing monensin, did not weaken the immune system of turkeys. The administration of monensin, in particular when combined with vaccination, was least effective in inhibiting inflammatory responses. Histological changes in immunocompetent organs (fatty degeneration) were also most severe in birds receiving monensin, followed by those administered doxycycline and enrofloxacin. The observed changes were exacerbated by vaccination.

Pathogenic bacteria associated with outbreaks of respiratory disease in Iranian broiler farms.

Multi-causal respiratory infections are more commonly observed than uncomplicated cases with single agents in the commercial poultry industry. Recently, increased mortality rates associated with respiratory clinical signs have been reported in Iranian broiler farms. The present study aimed to determine the spectra of avian mycoplasmas (Mycoplasma gallisepticum, MG and Mycoplasma synoviae, MS) and Ornithobacterium rhinotracheale (ORT) in the broiler farms with the multi-causal respiratory disease (MCRD) from 2017 to 2020. Trachea and lung tissue samples were collected from 70 broiler flocks presenting increased mortality and acute respiratory disease. MG, MS, and ORT were detected by performing polymerase chain reaction with primers complementary to the 16S rRNA, vlhA, and 16S rRNA genes, respectively. Genetic materials of MG, MS, and ORT were detected in five, three, and five of the 70 flocks. Based on the phylogenetic analysis of the complete mgc2 coding sequences, all MG strains formed a distinct cluster along with other Iranian MG isolates. According to the phylogenetic analysis of the partial vlhA gene of MS strains, two isolates were located along with Australian and European strains. In addition, one of them displayed an out-group association with MS isolates from Jordan. Phylogenetic analysis of Iranian ORT strains using a partial sequence of the 16S rRNA gene showed a distinct group among the other ORT strains. The results indicate that MG, MS, and ORT are not predominantly responsible for the MCRD. However, continuous monitoring of poultry flocks could be significant for obtaining valuable information related to different MG, MS, and ORT strains and designing effective control strategies.

A Multiplex PCR Method for Simultaneous Detection of Infectious Laryngotracheitis Virus and Ornithobacterium rhinotracheale

To date, many fluorescence- and gel-based multiplex polymerase chain reaction (PCR) assays have been developed for the simultaneous detection of multiple infectious agents of respiratory disease in poultry. However, PCR assays are not available for other important emerging respiratory bacteria, such as Ornithobacterium rhinotracheale (ORT). We aimed to fill this gap by establishing a new duplex PCR method for the simultaneous detection of infectious laryngotracheitis virus (ILTV) and ORT. Multiplex primer design software was used to select the compatible multiplex primer pairs. It was determined that an annealing temperature of 65 °C and an initial concentration of 2.5 pmol/µL for each primer set were the most suitable conditions for multiplex PCR. The assay was confirmed to be specific, as it only detected the target pathogens, even in the presence of six non-target agents. The limit of detection was up to 103 copies/µL of template DNA for both ILTV and ORT. In the screening of 304 field samples, 23, 88, and 44 were positive for both ILTV and ORT, solely for ILTV, and solely ORT, respectively.

Antibacterial properties of Allium sativum against Ornithobacterium rhinotracheale and Phyllanthus amarus extracts against Escherichia coli

Antibacterial activity of garlic extracts (Allium sativum) against Ornithobacterium rhinotracheale (ORT) and Phyllanthus amarus extracts against Escherichia coli (E. coli) were evaluated by agar diffusion assay. The results showed that ORT were sensitive to pure garlic extract and garlic extract from alcohol. The diluted extract inhibited ORT at moderate levels. Relating to E. coli, 250 mg/mL of Phyllanthus extract destroy the bacteria while minimal bactericidal concentration of doxycycline and amoxicillin against Escherichia coli were 16 μg/mL and 64 μg/mL, respectively. The results contribute to evaluate garlic extract and Phyllanthus extract as antibiotic alternatives in animal production.

Conjugation of Quantum Dot (CdS) with Antibodies for Identification of Ornithobacterium Rhinotracheale

Conjugation of Quantum Dot (CdS) with Antibodies for Identification of Ornithobacterium Rhinotracheale

Molecular characterization of Ornithobacterium rhinotracheale strains isolated from chickens in the Northwestern Iran.

Ornithobacterium rhinotracheale is the etiological agent of chickens and turkeys' respiratory diseases, reduction of eggs, growth retardation, and death. Present research aimed tocond uct the isolation, recognition, and molecular investigations of the bacterium in commercial broiler chicken flocks in East Azerbaijan province, Northwest of Iran, by the partial sequencing of 16S rRNA gene, ERIC‑PCR, and RAPD‑PCR with the OPG11 and M13 primers. We obtained 330 specimens from tracheal swabs of 33 slaughtered broiler flocks, of which we found 14 isolates (4.24%) of five flocks (15.15%) to be O. rhinotracheale. Typing by RAPD assay with the OPG11 primer, and ERIC‑PCR, classified the isolates in two types of 1 and 2 molecular patterns, most of which belonged to type 1. However, the M13 primer‑based RAPD technique was inappropriate for distinguishing and categorizing the isolates andgenerating all of them in the same pattern. In a phylogenetic analysis of O. rhinotracheale based on 16S rRNA sequences, the strains generated three clusters (I‑III), in which all of the studied isolates fell in one cluster (cluster I). Based on the results obtained from the RAPD and ERIC‑PCR assays, the genetic patterns of broiler‑chicken‑isolated O. rhinotracheale strains in Northwestern Iran had no significant differences.

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.

Value of an autogenous vaccine against Ornithobacterium rhinotracheale in turkey broilers on technical performances and antibiotic reduction

Value of an autogenous vaccine against Ornithobacterium rhinotracheale in turkey broilers on technical performances and antibiotic reduction

Characterization of Ornithobacterium rhinotracheale isolates by serotyping and infrared spectroscopy

Characterization of Ornithobacterium rhinotracheale isolates by serotyping and infrared spectroscopy