Molecular detection and pyrG sequence analysis of Avibacterium paragallinarum using clinical samples of infraorbital exudates from layer chickens with infectious coryza symptoms in Indonesia

Abstract

Background and Aim: Infectious coryza (IC) or snot, is caused by Avibacterium paragallinarum and leads to upper respiratory disease in poultry. Various diagnostic methods are available, including isolation and identification through bacterial culture and biochemical tests. However, the isolation and subsequent identification of A. paragallinarum are challenging because the bacteria are fastidious and require specific growth factors. This study aimed to detect A. paragallinarum in clinical samples taken from the exudate of the infraorbital sinus of layer hens showing clinical signs of IC. Materials and Methods: Samples were collected from 10 layer hens with IC symptoms. Following DNA extraction, HPG-2 polymerase chain reaction (PCR) assays were performed. The PCR amplicons underwent electrophoresis to determine those of the correct target size (511 bp), and these were sequenced. The resultant sequences were analyzed using the National Center for Biotechnology Information (NCBI) basic local alignment search tool. MEGA X was used for bioinformatics analysis. Results: The presence of A. paragallinarum was confirmed by HPG-2 PCR in 4/10 samples. Bioinformatics analysis showed that the amino acid sequence of the samples and the A. paragallinarum reference sequences in the NCBI database were grouped within the same cluster. Furthermore, the nucleotide sequences showed 98.64%–100% of similarity with the reference sequences. The phylogenetic reconstruction of partial pyrG sequences from 55 A. paragallinarum strains/isolates deposited in GenBank confirmed that the four HPG-2 PCR-positive samples fell within the A. paragallinarum cluster, separate from the Pasteurella multocida, Avibacterium spp., and Rodentibacter pneumotropicus clusters. Conclusion: Avibacterium paragallinarum infection was molecularly confirmed in 4/10 (40%) samples by HPG-2 PCR amplicon detection. Clustering of the pyrG partial gene sequences revealed that the positive samples fell within the A. paragallinarum cluster. Keywords: Avibacterium paragallinarum, bioinformatics, nucleotide sequencesm, polymerase chain reaction.