Eight complete and four draft genome sequences of nonpathogenic Avibacterium paragallinarum isolates from naive, healthy layer chickens in the USA

Article type: Brief Report

Keywords: nonpathogenic, naive, healthy layer chickens, infectious coryza

Authors: Mostafa M. S. Shelkamy, Amro Hashish ⚬, Mariela E. Srednik ⚬, Eman Gadu ⚬, Maria Chaves ⚬, Nubia Macedo ⚬, Qijing Zhang ⚬, Yuko Sato ⚬, Stephan Schmitz-Esser ⚬, Mohamed El-Gazzar ⚬

Affiliations: Department of Veterinary Diagnostic and Production Animal Medicine, College of Veterinary Medicine, Iowa State University1177https://ror.org/04rswrd78, Ames, Iowa, USA; Department of Avian and Rabbit Medicine, Faculty of Veterinary Medicine, Suez Canal University68831https://ror.org/02m82p074, Ismailia, Egypt; National Laboratory for Veterinary Quality Control on Poultry Production, Animal Health Research Institute, Agriculture Research Center357113https://ror.org/05xcgsy22, Giza, Egypt; Department of Veterinary Sciences, University of Wyoming4416https://ror.org/01485tq96, Laramie, Wyoming, USA; Department of Avian and Rabbit Diseases, Faculty of Veterinary Medicine, Mansoura University68779https://ror.org/01k8vtd75, Mansoura, Egypt; Department of Veterinary Microbiology and Preventive Medicine, College of Veterinary Medicine, Iowa State University1177https://ror.org/04rswrd78, Ames, Iowa, USA; Department of Animal Science, Iowa State University1177https://ror.org/04rswrd78, Ames, Iowa, USA

License: Copyright © 2025 Shelkamy et al. CC BY 4.0 This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.

Article links: DOI: 10.1128/mra.01334-24  |  PubMed: 40815000  |  PMC: PMC12424313

Relevance: Moderate: mentioned 3+ times in text

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ANNOUNCEMENT

Avibacterium paragallinarum (AP) is the causative agent of infectious coryza (IC) in chickens, leading to significant economic losses in the poultry industry (ref. 1). Recently, genetically divergent nonpathogenic AP (npAP) isolates, which still fall within the AP species, were identified in naive, healthy layer (NHL) flocks (ref. 2, ref. 3). npAP is prevalent in the US NHL flocks, complicating the diagnosis of IC (ref. 4).

In this study, bacterial isolates were obtained from the infraorbital sinuses of NHL chickens (ref. 5) using selective media consisting of Mueller-Hinton agar, fetal bovine serum, nicotinamide adenine dinucleotide, and inhibitors (ref. 6). Streaked plates were incubated for 48 hours at 37°C with 5.2% CO₂, and AP colonies were confirmed by MALDI-TOF (ref. 7) and the recN qPCR assay (ref. 8).

For Illumina sequencing, bacterial DNA was extracted using the KingFisher Flex machine and the MagMAX Pathogen RNA/DNA Kit (Thermo Fisher Scientific, Waltham, MA, USA). Sequencing libraries were prepared using different kits (Table 1) to generate 2 × 250 bp or 2 × 300 bp paired-end reads and sequenced on the Illumina MiSeq system (Illumina, San Diego, CA, USA). For Oxford Nanopore Technology (ONT) sequencing, genomic DNA extraction was performed using the Genomic-tip 20/G kit (Qiagen, Germany). ONT libraries were prepared using the Ligation Sequencing Kit (SQK-LSK109), barcoded with the Native Barcoding Expansion (EXP-NBD114), and DNA fragments of 3 kb or longer were enriched by Long Fragment Buffer from ONT (Oxford, UK). Sequencing was conducted on a MinION Mk1B flow cell R9.4.1 (FLO-MIN106) for 72 hours and monitored via MinKNOW (version 23.04.6). Post-run base calling was performed with Guppy (version 6.5.7) with super high accuracy mode enabled.

Default parameters were used for all software tools and web-based platforms unless otherwise indicated. Trim Galore version 0.6.5 (ref. 11) was used to trim low-quality bases and remove sequencing adaptors from the Illumina reads. The hybrid assembler Unicycler version 0.4.8 (ref. 12) was used to assemble two genomes, while the long-read assembler Flye version 2.9.1 (ref. 13) followed by two rounds of polishing with Illumina reads using Pilon version 1.23 (ref. 14) was used for the remaining 10 genomes (Table 1). The selection of either assembler was based on assembly quality as well as the quality and quantity of long-read data generated. Bandage plot version 0.8.1 (ref. 15) was used to check the circularity of chromosomes, and Quast version 5.2 (ref. 16) was used to assess assembly quality. All genomes were annotated using the RASTtk (ref. 17). These tools were accessed through BV-BRC version 3.35.5 (ref. 18). The publicly available assemblies in GenBank were annotated via PGAP version 6.8 (ref. 9).

While all 12 genomes were classified as npAP, they exhibited notable variability in key genes. For instance, four isolates are encapsulated (hctA+), whereas the others are not. Additionally, three npAP isolates display catalase positivity, differing from the typical biochemical activity of AP species. Two distinct putative plasmids were identified. Despite this variability, the lengthy HMTp210 gene, ranging from 12,876 to 20,046 bp due to unique insertions, remains a defining feature of npAP. A summary of data is shown in Table 1.

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