ABSTRACT
Ornithobacterium rhinotracheale has been associated with respiratory disease in poultry, particularly turkeys, leading to significant economic losses. However, O. rhinotracheale is poorly studied, and a very limited number of complete genomes are available. Here, we report the complete genome sequences of three O. rhinotracheale strains, generated using a Nanopore-Illumina hybrid assembly approach.
ANNOUNCEMENT
Ornithobacterium rhinotracheale has emerged as an important bacterial pathogen for domestic poultry and wild birds (1–10); however, it is an understudied pathogen. Only seven complete genomes are available in GenBank (https://www.ncbi.nlm.nih.gov/genome/browse/#!/prokaryotes/10734/) to date (November 2022), three of which are from this study. Here, we report the complete genome sequences of three O. rhinotracheale strains, generated using a Nanopore-Illumina hybrid assembly approach (11). These complete genomes will facilitate better understanding of this pathogen.
Three O. rhinotracheale isolates were selected as a diverse group of isolates obtained from the laboratory of K. Nagaraja at the University of Minnesota. As described previously (12), bacterial reculture was performed on blood agar (with 5% sheep blood), and the cultures were incubated under microaerophilic conditions at 37°C for 48 h. Isolates were identified and confirmed as O. rhinotracheale using real-time PCR as described previously (13). DNA extraction was performed using a MagMAX pathogen RNA/DNA kit (Thermo Fisher Scientific, Waltham, MA, USA) on a Kingfisher Flex instrument (Thermo Fisher Scientific). Extracted DNA was used for sequencing library preparation with an Illumina DNA preparation-tagmentation kit with IDT for Illumina DNA/RNA unique dual (UD) indexes, set A (Illumina, USA). The sequencing was performed using the Illumina MiSeq system (v3 reagent kit, with 2 × 300-bp paired-end reads).
DNA extraction for Nanopore sequencing was performed with the Nanobind CBB Big DNA kit (Circulomics, Baltimore, MD, USA) using the Gram-negative bacteria high molecular weight DNA extraction protocol. Nanopore libraries were prepared by multiplexing DNA extracts from the three isolates using the SQK-LSK109 and EXP-NBD104 kits (Oxford Nanopore Technologies, UK) according to the manufacturer's protocol. Barcodes 1, 3, and 7 from the EXP-NBD104 kit were used.
Raw reads from Illumina sequencing were first trimmed for quality and sequencing adapters using Trimmomatic v0.33 (14) with default parameters. NanoFilt v2.8.0 (15) was used with default parameters to quality filter Nanopore reads and filter out sequences <1,000 bp in length. Hybrid assemblies were performed using Unicycler v0.5.0 (16) with default parameters. All genomes were annotated using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP) v6.1 (17) as part of the RefSeq prokaryotic genome annotation project (BioProject accession number PRJNA224116). Reads generated with each sequencing platform, assembly statistics, annotation of the closed genomes, sequence typing, average nucleotide identity (ANI) values, and GenBank accession numbers are presented in Table 1. The hybrid assembly resulted in the formation of complete, closed circular genomes for the three isolates. However, the number of contigs for K223 is two due to the presence of a plasmid, which resulted in one closed contig for the chromosome and one closed contig for the plasmid.
TABLE 1.
Sequencing, annotation, and typing results for the sequenced strains
| Parametera | Data for O. rhinotracheale strain: |
||
|---|---|---|---|
| A171 | K223 | 97 | |
| Raw sequence reads | |||
| Illumina paired-end read length (nucleotides) | 300 | 300 | 300 |
| No. of Illumina reads used | 99,581 | 130,665 | 50,152 |
| Avg Illumina coverage (×) | 381 | 484 | 62 |
| No. of Nanopore reads | 27,383 | 21,287 | 6,593 |
| Avg Nanopore coverage (×) | 97.08 | 75.18 | 97.12 |
| Nanopore read N50 (bp) | 15,541 | 17,237 | 2,188 |
| Assembly statistics for closed genomes | |||
| No. of contigs | 1 | 2 | 1 |
| Total chromosome length (bp) | 2,409,995 | 2,407,355 | 2,418,866 |
| Plasmidb | Absent | Present | Absent |
| Total plasmid length (bp) | NA | 14,785 | NA |
| GC content (%) | 37.45 | 37.37 | 37.43 |
| Hybrid assembly N50 (bp) | 2,409,995 | 2,407,355 | 2,418,866 |
| Annotation results | |||
| No. of CDSs (protein) | 2,237 | 2,289 | 2,247 |
| No. of noncoding RNAs | 3 | 3 | 3 |
| No. of tRNAs | 43 | 43 | 43 |
| No. of rRNAs | 9 | 9 | 9 |
| No. of CDSs (without protein) | 26 | 29 | 29 |
| No. of CRISPR arrays | 1 | 1 | 1 |
| No. of hypothetical proteins | 541 | 563 | 568 |
| No. of proteins with functional assignments | 1,697 | 1,726 | 1,679 |
| Serotype from agar gel precipitation test | A | K | Unknown |
| ST from O. rhinotracheale MLSTc | ST-1 | NA (too divergent to conclude ST) | ST-1 |
| ANIb (%) vs O. rhinotracheale type strain DSM 15997 (accession no. CP003283.1)d | 99.9 | 89.49 | 99.86 |
| GenBank accession no. | |||
| BioSample accession no. | SAMN27124716 | SAMN27139539 | SAMN27150225 |
| BioProject accession no. | PRJNA821850 | PRJNA821858 | PRJNA821870 |
| Complete genome assembly accession no. | |||
| Chromosome | CP094844 | CP094846 | CP094845 |
| Plasmid | CP094847 | ||
CDS, coding sequence; ST, sequence type; MLST, multilocus sequence typing; ANI, average nucleotide identity; NA, not applicable.
The presence or absence of plasmids in the genome sequences was determined using the Plasmid database (19) via PLSDB (https://ccb-microbe.cs.uni-saarland.de/plsdb).
MLST was performed using the O. rhinotracheale MLST scheme (20) available via PubMLST (21) (https://pubmlst.org/organisms/ornithobacterium-rhinotracheale).
ANIb are ANI values based on BLAST+ calculated via the JSpeciesWS online service (22) (https://jspecies.ribohost.com/jspeciesws/#home).
It is noteworthy (as shown in Table 1) that the ANI values for two genomes (A171 and 97) versus the O. rhinotracheale type strain DSM 15997 (accession number CP003283.1) were above the species cutoff value of 95 to 96%, while the ANI value for genome K223 was below the species cutoff value (89.49%). These findings support the suggestion by Alispahic et al. (18) that some O. rhinotracheale serotypes belong to a different bacterial species.
Data availability.
The genomes are available in GenBank under the accession numbers shown in Table 1.
ACKNOWLEDGMENTS
This project was supported by the Agriculture and Food Research Initiative (competitive grant 2015-68004-23131) from the U.S. Department of Agriculture, National Institute of Food and Agriculture. Bioinformatic analyses were supported with tools available from the Minnesota Supercomputing Institute of the University of Minnesota.
We thank the Iowa State University Veterinary Diagnostic Laboratory faculty and staff for assistance with sample processing and diagnostic testing.
Contributor Information
Mohamed El-Gazzar, Email: elgazzar@iastate.edu.
Catherine Putonti, Loyola University Chicago.
REFERENCES
- 1.Vandamme P, Segers P, Vancanneyt M, van Hove K, Mutters R, Hommez J, Dewhirst F, Paster B, Kersters K, Falsen E. 1994. Ornithobacterium rhinotracheale gen. nov., sp. nov., isolated from the avian respiratory tract. Int J Syst Bacteriol 44:24–37. doi: 10.1099/00207713-44-1-24. [DOI] [PubMed] [Google Scholar]
- 2.Clark S, Ahlmeyer V. 2018. Current health and industry issues facing the turkey industry. In The Proceeding of Annual meeting of the United States Animal Health Association 2018. United States Animal Health Association, St. Joseph, MO. [Google Scholar]
- 3.Al-Hasan BA, Alhatami AO, Abdulwahab HM, Bustani GS, Wahab Alkuwaity EA. 2021. The first isolation and detection of Ornithobacterium rhinotracheale from swollen head syndrome-infected broiler flocks in Iraq. Vet World 14:2346–2355. doi: 10.14202/vetworld.2021.2346-2355. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Canal CW, Leão JA, Rocha SLS, Macagnan M, Lima-Rosa CAV, Oliveira SD, Back A. 2005. Isolation and characterization of Ornithobacterium rhinotracheale from chickens in Brazil. Res Vet Sci 78:225–230. doi: 10.1016/j.rvsc.2004.10.003. [DOI] [PubMed] [Google Scholar]
- 5.Chávez M, León YM, Ugalde YS, Cárdenas NB, López AM, Rivero EL, Redondo AV. 2011. Evidência sorológica, em Cuba, da circulação de Ornithobacterium rhinotracheale em galinhas poedeiras com síndrome respiratória crônica. In XXII Congresso Latino-Americano de Avicultura. Latin America Association of Poultry. https://pt.engormix.com/avicultura/artigos/ornitobacterium-rhinotracheale-galinhas-poedeiras-t37346.htm. [Google Scholar]
- 6.Churria CDG, Sansalone P, Machuca M, Vigo G, Sguazza G, Origlia J, Píscopo M, Loyola MH, Petruccelli M. 2012. Tracheitis in a broiler chicken flock caused by dual infection with Cryptosporidium spp. (Apicomplexa: Cryptosporiidae) and non-hemolytic Ornithobacterium rhinotracheale. Braz J Vet Pathol 5:89–93. [Google Scholar]
- 7.Ha HJ, Christensen N, Humphrey S, Haydon T, Bernardi G, Rawdon T. 2016. The first detection of Ornithobacterium rhinotracheale in New Zealand. Avian Dis 60:856–859. doi: 10.1637/11457-062116-Case. [DOI] [PubMed] [Google Scholar]
- 8.Nisar M, Thieme S, Hafez HM, Sentíes-Cúe G, Chin RP, Muhammad SP, Aboubakr H, Goyal SM, Nagaraja KV. 2020. Genetic diversity of Ornithobacterium rhinotracheale isolated from chickens and turkeys in the United States. Avian Dis 64:324–329. doi: 10.1637/aviandiseases-D-20-00007. [DOI] [PubMed] [Google Scholar]
- 9.Umali DV, Shirota K, Sasai K, Katoh H. 2018. Characterization of Ornithobacterium rhinotracheale from commercial layer chickens in eastern Japan. Poult Sci 97:24–29. doi: 10.3382/ps/pex254. [DOI] [PubMed] [Google Scholar]
- 10.Van Veen L, Van Empel P, Fabri T. 2000. Ornithobacterium rhinotracheale, a primary pathogen in broilers. Avian Dis 44:896–900. doi: 10.2307/1593063. [DOI] [PubMed] [Google Scholar]
- 11.Goldstein S, Beka L, Graf J, Klassen JL. 2019. Evaluation of strategies for the assembly of diverse bacterial genomes using MinION long-read sequencing. BMC Genomics 20:23. doi: 10.1186/s12864-018-5381-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12.Hashish A, Sinha A, Sato Y, Macedo NR, El-Gazzar M. 2022. Development and validation of a new TaqMan real-time PCR for the detection of Ornithobacterium rhinotracheale. Microorganisms 10:341. doi: 10.3390/microorganisms10020341. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13.Hashish A, Sinha A, Sato Y, Macedo NR, El-Gazzar M. 2022. Correction: Hashish et al. Development and validation of a new TaqMan real-time PCR for the detection of Ornithobacterium rhinotracheale. Microorganisms 2022, 10, 341. Microorganisms 10:917. doi: 10.3390/microorganisms10050917. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14.Bolger AM, Lohse M, Usadel B. 2014. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30:2114–2120. doi: 10.1093/bioinformatics/btu170. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15.De Coster W, D'Hert S, Schultz DT, Cruts M, Van Broeckhoven C. 2018. NanoPack: visualizing and processing long-read sequencing data. Bioinformatics 34:2666–2669. doi: 10.1093/bioinformatics/bty149. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 16.Wick RR, Judd LM, Gorrie CL, Holt KE. 2017. Unicycler: resolving bacterial genome assemblies from short and long sequencing reads. PLoS Comput Biol 13:e1005595. doi: 10.1371/journal.pcbi.1005595. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 17.Tatusova T, DiCuccio M, Badretdin A, Chetvernin V, Nawrocki EP, Zaslavsky L, Lomsadze A, Pruitt KD, Borodovsky M, Ostell J. 2016. NCBI Prokaryotic Genome Annotation Pipeline. Nucleic Acids Res 44:6614–6624. doi: 10.1093/nar/gkw569. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 18.Alispahic M, Endler L, Hess M, Hess C. 2021. Ornithobacterium rhinotracheale: MALDI-TOF MS and whole genome sequencing confirm that serotypes K, L and M deviate from well-known reference strains and numerous field isolates. Microorganisms 9:1006. doi: 10.3390/microorganisms9051006. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 19.Schmartz GP, Hartung A, Hirsch P, Kern F, Fehlmann T, Müller R, Keller A. 2022. PLSDB: advancing a comprehensive database of bacterial plasmids. Nucleic Acids Res 50:D273–D278. doi: 10.1093/nar/gkab1111. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 20.Thieme S, Mühldorfer K, Lüschow D, Hafez HM. 2016. Molecular characterization of the recently emerged poultry pathogen Ornithobacterium rhinotracheale by multilocus sequence typing. PLoS One 11:e0148158. doi: 10.1371/journal.pone.0148158. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 21.Jolley KA, Bray JE, Maiden MCJ. 2018. Open-access bacterial population genomics: BIGSdb software, the PubMLST.org website and their applications. Wellcome Open Res 3:124. doi: 10.12688/wellcomeopenres.14826.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 22.Richter M, Rosselló-Móra R, Oliver Glöckner F, Peplies J. 2016. JSpeciesWS: a web server for prokaryotic species circumscription based on pairwise genome comparison. Bioinformatics 32:929–931. doi: 10.1093/bioinformatics/btv681. [DOI] [PMC free article] [PubMed] [Google Scholar]
Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
The genomes are available in GenBank under the accession numbers shown in Table 1.