Fowl cholera caused by Pasteurella multocida type A poses significant challenges for poultry production. This study collected 80 organ samples from commercial layer farms in Bangladesh and applied cultural, biochemical, and molecular methods to identify P. multocida. Twelve isolates (15%) were confirmed as type A. Antimicrobial susceptibility testing to 16 agents revealed resistance to cefixime, ceftriaxone, ertapenem, and meropenem, with highest sensitivity to ciprofloxacin, doxycycline, tetracycline, and levofloxacin. A representative isolate was sequenced on an Illumina MiSeq platform. RAST annotation reported a genome of 2,346,689 bp, 2,205 coding sequences, 59 RNA genes, and GC content of 40.2%. Virulence-associated genes, a high PathogenFinder probability (0.891) for human pathogenicity, and phylogenetic clustering with strains from Australia, China, and Malaysia were observed. These genomic and phenotypic data provide baseline information for surveillance and targeted antimicrobial selection in fowl cholera control.
Commercial poultry in Bangladesh has expanded rapidly and is economically important for nutrition and livelihoods. Despite this growth, infectious diseases continue to cause production losses. Fowl cholera (FC), an acute septicemic disease, is caused by P. multocida and remains a critical threat, with reported prevalence varying across flock types. P. multocida is classified into capsular types A, B, D, E, and F; in Asia, types A:1, A:3, and D are commonly implicated in FC. Traditional phenotypic and molecular tools are routinely used for outbreak response but can lack genomic resolution. Whole-genome sequencing offers comprehensive insight into virulence, antimicrobial resistance (AMR), host adaptation, and phylogeny. Prior to this work, no whole-genome sequence data for P. multocida type A from Bangladeshi layer hens were available.
Samples were obtained from commercial layer farms in Trishal, Valuka, and Gazipur. Each sampled farm housed over 1,000 hens; five hens per farm were sampled, yielding 80 hens in total. From each bird, heart, liver, and spleen tissues were collected from birds that had died or were clinically suspected of fowl cholera. Samples were transported under cold-chain conditions to the Department of Microbiology and Hygiene at Bangladesh Agricultural University.
Tissue specimens were inoculated into nutrient broth and incubated aerobically at 37 °C. Growth-positive broths were streaked onto nutrient agar and 5% bovine blood agar for enrichment; MacConkey agar was used for further confirmation. Colonies were examined by Gram stain and subjected to biochemical tests including MR-VP, indole, catalase, oxidase, and carbohydrate fermentation (dextrose, maltose, lactose, mannitol, sucrose). Pure isolates were preserved in 70% glycerol stocks or on blood agar slants under refrigeration.
Molecular confirmation used PCR targeting the KMT1 and capA genes. DNA templates were prepared by heat lysis of colonies and PCR reactions were performed using standard master mix and primer concentrations as described in the methods.
Antimicrobial susceptibility testing employed the disc diffusion method against a panel of 16 antimicrobials. A representative isolate was selected for whole-genome sequencing on the Illumina MiSeq platform, and genomic analysis was performed using multiple bioinformatics tools including RAST for annotation and MEGA X for phylogenetic analysis.
From 80 suspected FC samples, P. multocida was confirmed in 12 isolates (15%) and typed as capsular type A by PCR. Antimicrobial susceptibility testing showed phenotypic resistance to cefixime, ceftriaxone, ertapenem, and meropenem. The greatest susceptibility was observed for ciprofloxacin, doxycycline, tetracycline, and levofloxacin.
Whole-genome sequencing of a representative isolate produced an assembly annotated by RAST with a genome size of 2,346,689 bp, 2,205 predicted coding sequences, 59 RNA genes, and a GC content of 40.2%. Bioinformatic screening identified multiple virulence-associated genes, including wecA, galU, manB, rfaD, rfaE, rfaF, lpxB, lpxC, and msbA, indicative of lipopolysaccharide-related functions and potential roles in immune evasion.
PathogenFinder analysis predicted the isolate as a potential human pathogen with a probability score of 0.891. Phylogenetic analysis using MEGA X and the maximum likelihood method placed the isolate within a monophyletic clade and indicated closest genetic relatedness to published strains from Australia, China, and Malaysia.
This investigation combines phenotypic and genomic approaches to characterize P. multocida type A from layer hens. The isolation rate in this sample set was 15%. The antibiogram reveals resistance to several beta-lactams and carbapenems tested, and retained susceptibility to fluoroquinolones and tetracyclines, which has implications for empirical therapy and stewardship in poultry. WGS provided detailed genomic context: assembly metrics and annotation quantify coding capacity and RNA content, and identified virulence loci related to lipopolysaccharide biosynthesis and transport.
The PathogenFinder probability suggests zoonotic potential, and the phylogenetic relationships to strains from other countries underscore international genetic links or shared lineages. These genomic data fill a local knowledge gap and may explain partial vaccine failures or variable control outcomes when vaccines lack representation of circulating genomic features.
Limitations include that only one isolate was subjected to WGS in this report; the study does not report detailed clinical histories, flock-level performance, or longitudinal sampling. The authors note that additional genomic surveillance would strengthen outbreak investigation and vaccine strain selection.
This study provides the first whole-genome sequence and antibiogram data for P. multocida type A from Bangladeshi layer hens. Key findings include a 15% isolation rate among sampled birds, resistance to certain cephalosporins and carbapenems, susceptibility to fluoroquinolones and tetracyclines, a 2.35 Mbp genome with over 2,200 coding sequences, identified virulence genes, a high PathogenFinder probability for human pathogenicity, and phylogenetic proximity to strains from Australia, China, and Malaysia. These results offer baseline genomic and phenotypic information to inform surveillance, targeted antimicrobial selection, and future vaccine and control strategy development.
The whole genome shotgun sequencing data for the representative isolate are publicly available in NCBI under BioProject JAODLP000000000.1. The authors stated they will provide other requested information on request.